Biological materials related to HER3

ABSTRACT

The present disclosure relates to amino acid sequences that are directed against (as defined herein) HER3, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively). The disclosure also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for 10 preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is the National Phase of International PatentApplication No. PCT/FP2011/058295, tiled May 20, 2011, published as WO2011/144749, which claims priority to U.S. Provisional Application No.61/346,548, filed May 20, 2010. The contents of these applications areherein incorporated by reference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 23, 2016, isnamed 103926-0100_SL.txt and is 840,697 bytes in size.

The present invention relates to amino acid sequences that are directedagainst (as defined herein) HER3, as well as to compounds or constructs,and in particular proteins and polypeptides, that comprise oressentially consist of one or more such amino acid sequences (alsoreferred to herein as “amino acid sequences of the invention”,“compounds of the invention”, and “polypeptides of the invention”,respectively).

In some specific, but non-limiting aspects (described in more detailherein), the invention provides:

-   -   amino acid sequences that are directed against (as defined        herein) HER3 and that are capable of inhibiting or blocking        (fully or partially, as further described herein) the binding of        HRG to HER3 (as further described herein);    -   amino acid sequences that are directed against (as defined        herein) HER3 and that are capable of inhibiting or blocking        (fully or partially, as further described herein)        heterodimerization of HER3 (as further described herein);    -   amino acid sequences that are directed against (as defined        herein) HER3 and that are capable of binding to domain II of        HER3; and/or    -   amino acid sequences that are directed against (as defined        herein) HER3 and that are capable of inhibiting or blocking        (fully or partially, as further described herein) HER3        phosphorylation (as further described herein).

As further described herein, in a specific preferred, but non-limitingaspect, the various amino acid sequences directed against HER3 that aredescribed herein (including those according to specific aspects) arepreferably immunoglobulin single variable domains (also referred toherein as “ISV's). An immunoglobulin single variable domain is an aminoacid sequence that:

-   -   comprises an immunoglobulin fold or that, under suitable        conditions (such as physiological conditions) is capable of        forming an immunoglobulin fold (i.e. by folding), i.e. so as to        form an immunoglobulin variable domain (such as, for example, a        VH, VL or VHH domain);

and that

-   -   forms (or under such suitable conditions is capable of forming)        an immunoglobulin variable domain that comprises a functional        antigen binding activity (in the sense that it does not require        an interaction with another immunoglobulin variable domain (such        as a VH-VL interaction) to form a functional antigen binding        site).

Amino acid sequences of the invention that are ISV's are also referredto herein as “ISV's of the invention”. Some preferred examples ofimmunoglobulin single variable domains suitable for use in the inventionwill become clear from the further description herein, and for examplecomprise VHH's and/or (other) Nanobodies (preferred) such as humanizedVHH's or camelized VH's such as camelized human VH, dAb's and (single)domain antibodies.

As also further described herein, the various amino acid sequencesdirected against HER3 that are described herein (including thoseaccording to specific aspects) can with advantage be used as buildingblocks to provide multivalent (as described herein, such as bi- ortrivalent), multispecific (as described herein, such as bi- ortrispecific) or multiparatopic (as described herein, such asbiparatopic) polypeptides of the invention, and such polypeptides of theinvention for further preferred but non-limiting aspects of theinvention. Again, also in these aspects of the invention, the amino acidsequences of the invention present in such polypeptides are preferablyISV's (and preferably nanobodies as described herein).

For example and without limitation, in one specific aspect of theinvention, such a polypeptide may comprise at least one (such as one ortwo) ISV's (and preferably nanobodies) that are directed against (asdefined herein) HER3 and that are capable of inhibiting or blocking(fully or partially, as further described herein) the binding of HRG toHER3 (as further described herein) and at least one (such as one or two)ISV's (and preferably nanobodies) that are directed against (as definedherein) HER3 and that are capable of inhibiting or blocking (fully orpartially, as further described herein) heterodimerization of HER3 (asfurther described herein). These and the other amino acid sequences andpolypeptides of the invention may also have been provided with anincreased half-life in vivo, as further described herein.

Some preferred but non-limiting examples of the various amino acidsequences and polypeptides of the invention will become clear from thefurther description herein.

The invention also relates to nucleic acids encoding such amino acidsequences and polypeptides (also referred to herein as “nucleic acids ofthe invention” or “nucleotide sequences of the invention”); to methodsfor preparing such amino acid sequences and polypeptides; to host cellsexpressing or capable of expressing such amino acid sequences orpolypeptides; to compositions, and in particular to pharmaceuticalcompositions, that comprise such amino acid sequences, polypeptides,nucleic acids and/or host cells; and to uses of such amino acidsequences or polypeptides, nucleic acids, host cells and/orcompositions, in particular for prophylactic, therapeutic or diagnosticpurposes, such as the prophylactic, therapeutic or diagnostic purposesmentioned herein.

Other aspects, embodiments, advantages and applications of the inventionwill become clear from the further description herein.

BACKGROUND OF THE INVENTION

HER3 (human epidermal growth factor receptor 3) belongs to the ErbB/HERsubfamily of polypeptide growth factor receptors, which includes theepidermal growth factor (EGF) receptor (EGFR, ErbB1, HER1), the neuoncogene product (ErbB2, HER2), and the more recently identified ErbB3,HER3 and ErbB4, HER4 receptor proteins (see, e.g., Plowman et al.(1990), Proc. Natl. Acad. Sci. USA 87, 4905-4909; Hynes et. al. (1994)Biochim. Biophys. Acta Rev. Cancer 1198, 165-184). It is known that HER3can bind multiple ligands, such as heregulin and the neuregulins 1 and2; but that it lacks intrinsic tyrosine kinase activity (and because itis kinase inactive, the receptor can only initiate signal transductionwhen dimerized with another HER family member, such as HER1, HER2 orHER4).

More specifically, HER3 is a membrane-bound protein and has a neuregulinbinding domain but not an active kinase domain. It therefore can bindthis ligand but not convey the signal into the cell through proteinphosphorylation. However, it does form heterodimers with other EGFreceptor family members which do have kinase activity.Heterodimerization leads to the activation of pathways which lead tocell devision, proliferation, differentiation, migration and othercellular processes. Complex multilayered signaling generated receptorcross-talk and lateral signaling is becoming evident within the EGFRfamily and other receptor tyrosine kinases like MET (Engelmann et. al.(2007), Science 316, 1039-1043). Deregulated, aberrant signaling due tomutation, amplification and presence of active autrocrine loops mayparticipate in development of cancer and other diseases. Amplificationof this gene and/or overexpression of its protein have been reported innumerous cancers, including prostate, bladder, and breast tumors (seee.g. WO2008100624, WO2007077028, Horst et al. (2005) Int J Cancer, 115,519-527; Xue et al. (2006) Cancer Res. 66, 1418-1426). HER3 is alsoknown as LCCS2; ErbB-3; c-erbB3; erbB3-S; MDA-BF-1; MGC88033; c-erbB-3;p180-ErbB3; p45-sErbB3; p85-sErbB3; ERBB3.

When it comes to the role of HER3 in cancer, it has been suggested thatHER3 may be necessary for HER2-mediated tumorigenesis, in the sense thatHER2 may require HER3 in order to transform normal cells into cancercells. For example, it has been found that increased expression of HER3increases the signaling potency of HER2, whereas decreased HER3expression results in the loss of HER2 activity. This has led to thehypothesis that HER3 may be involved in HER2-mediated tumorigenesisthrough dimerization with HER2.

It has also been suggested that HER3 may enable escape from inhibitionof other HER receptors. For example, preclinical research has shown thatupregulation of HER3 activity may be a mechanism by which tumor cellscan escape tyrosine kinase inhibition of HER family receptors, and thattumor cells may compensate for tyrosine kinase inhibition of other HERreceptors by increasing expression of HER3, which is kinase inactive. Ithas also been found that in HER2:HER3 heterodimers, HER2transphosphorylates HER3.

HER3 has also been found to be overexpressed in several types of cancer(including without limitation breast and pancreatic cancer), and it hasbeen found that there may be a correlation between the expression ofHER2/HER3 and the progression from an non-invasive to an invasive stage(Baselga et al., 2009 Nature Reviews Cancer 9, 463-475).

Although, the role of HER3 in cancer and oncogenic signaling has beenimplicated (supra), its importance in an anti-cancer treatment remainsunclear due to the complex ErbB network in which HER3 is a componentthereof. Current immunotherapies primarily focus on inhibiting theaction of HER2 and, in particular, heterodimerization of HER2/HER3complexes (see, e.g., Sliwkowski et al. (1994) J. Biol. Chem. 269(20):14661-14665).

It is an object of the present invention to provide improvedimmunotherapies that effectively inhibit HER3 signaling, and can be usedto treat and diagnose a variety of cancers.

SUMMARY OF THE INVENTION

In the invention, a number of immunoglobulin single variable domains (asfurther described herein) have been identified and characterized (andwhere appropriate humanized and/or sequence optimized) that can bind toHER3 (and in particular specifically bind to HER3, as further definedherein) and that can (be used to) modulate (as defined herein) HER3mediated signalling and/or modulate (some or all of) the biologicaleffects of HER3 and/or modulate (some or all of) the biologicalmechanisms/pathways in which HER3 and/or HER3 mediated signalling isinvolved. It has also been found that the immunoglobulin single variabledomains provided by the present invention can not only be used per sefor such modulation, but can also with further advantage be linkedto/combined with each other (i.e. so as to provide multivalent,multispecific and/or biparatopic constructs, as further defined herein)and/or with other moieties, binding domains or binding units to provideproteins, polypeptides or (other) compounds or constructs that can beused for such modulation.

Thus, the use of the immunoglobulin single variable domains (or “ISV's”)provided by the invention as “building blocks” for providing suchproteins, polypeptides or (other) compounds or constructs forms animportant advantage and aspect of the invention.

For example, and with advantage but without limitation, it has beenfound that the various immunoglobulin single variable domains or “ISV's”provided by the invention can bind in different ways to HER3, and thusprovide different modes-of-action in the way that they interact withHER3 and/or modulate HER3 mediated signalling. For example, some of theISV's provided by the invention are capable of inhibiting binding of HRGto HER3, whereas others can bind to domain II of HER3 and/or block HER3transphosphorylation. Yet others can block dimerisation of HER3 withitself of with other members of the HER family (such as HER1, HER2 orHER3). Hence, the amino acid of the invention (or ISV) is considered abuilding block.

Thus, the invention provides a range of different ISV's that caninfluence HER3, HER3 mediated signalling and/or the biological effectsassociated with HER3 and/or with HER3 mediated signalling.

In addition, when the ISV's provided by the invention are suitably usedor combined as building blocks to provide further proteins, polypeptidesor (other) compounds or constructs, the invention for example makes itpossible, with advantage, to combine different interactions with HER3and/or different mode-of-actions into a single molecule, compound,construct, protein or polypeptides. Examples of the same will becomeclear from the further description herein.

The effects or influence that the various ISV's, proteins, polypeptides,compounds and/or constructs that are provided by the invention have onHER3 and HER3 mediated signalling (including their mode/modes of action)can be determined using various suitable assays and in vivo models, suchas an HER3 internalization assay (for example measuring reduction HER3surface expression on a suitable cell such as MCF7 and MALME-3M cells);ligand blocking assays (such as HRG competition Alphascreen or FACSassays); HRG induced HER3 signalling blocking assays (such as an assaymeasuring inhibition of pHER3 in HER3-ligand stimulated MCF7,CHO-HER2-HER3, BT474 and MDA-MB468 cells); assays measuringheterodimerization blocking (such as pHER3 blocking ofTGF-alpha-stimulated CHO-EGFR-HER3, β-cellulin-stimulated MDA-MB468cells); assay measuring inhibition of downstream signalling (such as anassay measuring pAKT and/or pMAPK signaling in HER3-ligand stimulatedMCF7, A549 and BT474 cells); or cell migration assays (such as assaysmeasuring HRG induced migration of A431 cells). Reference is for examplemade to the Experimental Section and the results presented therein.

Thus, the ISV's, polypeptides and compositions of the present inventioncan generally be used to modulate, and in particular inhibit and/orprevent, binding of HER3 to Heregulin and/or blocking heterodimerizationwith MET, EGFR or HER2 (see e.g. Hsieh and Moasser, 2007, BritishJournal of Cancer), and thus to modulate, and in particular inhibit orprevent, the signalling that is mediated by HER3 and/or Heregulin, tomodulate the biological pathways in which HER3 and/or Heregulin areinvolved, and/or to modulate the biological mechanisms, responses andeffects associated with such signalling or these pathways.

As such, the polypeptides and compositions of the present invention canbe used for the diagnosis and treatment (as defined herein) of a varietyof cancers. Generally, “variety of cancers” can be defined as diseasesand disorders that can be prevented and/or treated, respectively, bysuitably administering to a subject in need thereof (i.e. having thedisease or disorder or at least one symptom thereof and/or at risk ofattracting or developing the disease or disorder) of either apolypeptide or composition of the invention (and in particular, of apharmaceutically active amount thereof) and/or of a known activeprinciple active against HER3 or a biological pathway or mechanism inwhich HER3 is involved (and in particular, of a pharmaceutically activeamount thereof). Examples of such variety of cancers will be clear tothe skilled person based on the disclosure herein, and for exampleinclude the following diseases and disorders:

Cancer (Sithanandam and Anderson review (2008) Cancer Gene Therapy15(7), 413-448; breast cancer (Lemoine et. al. (1992) Br J Cancer 66,1116-1121; Witton et al. (2003) J Pathol 200(3):290-297; Koutras et al(2010) Crit. Rev Oncol Hematol. 74(2):73-78); lung cancer (Müller-Tidow(2005) Cancer Res 65(5):1778-1782; Timotheadou et al., (2007) AnticancerRes. 27(6C):4481-4489); ovarian cancer (Tanner et. al. (2006) J ClinOncol 24(26): 4317-4323); prostate cancer (Lozano et. al. (2005) BMCGenomics 6:109; Soler et al., 2009. Int J Cancer 125(11):2565-2575)urinary bladder cancer (Rajkumar et. al. (1996) J Pathol,179(4):381-385); brain cancer (Addo-Yobo et. al. (2006) J NeuropatholExp Neurol 65(8):769-775, Andersson et. al. (2004) Acta Neuropathol,108(2):135-142); Retinoblastoma (Chakraborty et. al. (2007) Genomics90(3):344-353); melanoma (Segal et. al. (2003) J Clin Oncol. 2003 May 1;21(9):1775-1781; Schaefer et. al. (2004) Cancer Res 64:3395-3405;Reschke et al., 2008 Clin Cancer Res. 14(16):5188-97); colorectal cancer(Grivas et. al. (2007) Eur J Cancer 43(17):2602-2611; Ciardiello et al.(1991) Proc Natl Acad Sci USA. 88(17):7792-7796); pancreatic cancer(Friess et. al. (1995) Clin Cancer Res 1(11):1413-20); Lemoine et al.,1992 J. Pathol. 168: 269-273); gastric cancer (Sanidas (1993), Int JCancer 54(6):935-40, Hayashi et. al. (2008) Clin Cancer Res14(23):7843-7849; Hayashi et al., (2008) Clin Cancer Res.14(23):7843-9); head and neck cancer (Funayama (1998) Oncology55(2):161-167, Erjala (2006) Clin Cancer Res 12(13):4103-4111); cervixcancer (Fuchs et al., 2007 Anticancer Res. 27(2):959-63); oesophaguscancer (Wei et al., 2007 Int J. Oncol. 31(3):493-9.); and/or nerveregeneration (Lindholm et. al. (2002) Exp Brain Res) 2002 January;142(1):81-90.

In particular, the polypeptides and compositions of the presentinvention can be used for the diagnosis and treatment of variety ofcancers which are characterized by excessive and/or unwanted signallingmediated by ErbB network of proteins or in general by any pathway(s) inwhich HER3 is involved. Examples of such variety of cancers will againbe clear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, it is also envisaged that thepolypeptides of the invention can be used to prevent and/or to treat alldiseases and disorders for which treatment with such active principlesis currently being developed, has been proposed, or will be proposed ordeveloped in future. In addition, it is envisaged that, because of theirfavourable properties as further described herein, the polypeptides ofthe present invention may be used for the prevention and treatment ofother diseases and disorders than those for which these known activeprinciples are being used or will be proposed or developed; and/or thatthe polypeptides of the present invention may provide new methods andregimens for treating the diseases and disorders described herein.

Other applications and uses of the amino acid sequences and polypeptidesof the invention will become clear to the skilled person from thefurther disclosure herein.

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the diagnosis, prevention and/or treatment of a variety ofcancers and of the further diseases and disorders mentioned herein; andto provide methods for the diagnosis, prevention and/or treatment ofsuch diseases and disorders that involve the administration and/or useof such agents and compositions.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions and/or methods that havecertain advantages compared to the agents, compositions and/or methodsthat are currently used and/or known in the art. These advantages willbecome clear from the further description below.

More in particular, it is an object of the invention to providetherapeutic proteins that can be used as pharmacologically activeagents, as well as compositions comprising the same, for the diagnosis,prevention and/or treatment of variety of cancers and of the furtherdiseases and disorders mentioned herein; and to provide methods for thediagnosis, prevention and/or treatment of such diseases and disordersthat involve the administration and/or the use of such therapeuticproteins and compositions.

Accordingly, it is a specific object of the present invention to provideamino acid sequences that are directed against (as defined herein) HER3,in particular against HER3 from a warm-blooded animal, more inparticular against HER3 from a mammal, and especially against human HER3(SEQ ID NO: 1); and to provide proteins and polypeptides comprising oressentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat are suitable for prophylactic, therapeutic and/or diagnostic use ina warm-blooded animal, and in particular in a mammal, and more inparticular in a human being.

More in particular, it is a specific object of the present invention toprovide such amino acid sequences and such proteins and/or polypeptidesthat can be used for the prevention, treatment, alleviation and/ordiagnosis of one or more diseases, disorders or conditions associatedwith HER3 and/or mediated by HER3 (such as the diseases, disorders andconditions mentioned herein) in a warm-blooded animal, in particular ina mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acidsequences and such proteins and/or polypeptides that can be used in thepreparation of pharmaceutical or veterinary compositions for theprevention and/or treatment of one or more diseases, disorders orconditions associated with and/or mediated by HER3 (such as thediseases, disorders and conditions mentioned herein) in a warm-bloodedanimal, in particular in a mammal, and more in particular in a humanbeing.

In the invention, generally, these objects are achieved by the use ofthe amino acid sequences, proteins, polypeptides and compositions thatare described herein.

In general, the invention provides amino acid sequences that aredirected against (as defined herein) and/or can specifically bind (asdefined herein) to HER3; as well as compounds and constructs, and inparticular proteins and polypeptides, that comprise at least one suchamino acid sequence.

As already mentioned, in some specific, but non-limiting aspects(described in more detail herein), the invention provides:

amino acid sequences that are directed against (as defined herein) HER3and that are capable of inhibiting or blocking (fully or partially, asfurther described herein) ligand binding, and in particular ofinhibiting or blocking (fully or partially, as further described herein)the binding of HRG to HER3 (as further described herein). These aminoacid sequences are also referred to herein as “HRG-blocking amino acidsequences” or “HRG-blocking building blocks”. Preferably, theseHRG-blocking amino acid sequences are ISV's (as described herein), inwhich case they are also referred to as “HRG-blocking ISV's”.Preferably, any HRG-blocking amino acid sequences, HRG-blocking buildingblocks or HRG-blocking ISV's are such that they have blocking activity,i.e. block HRG binding to HER3 partially or completely, which can bedetermined by any suitable assay known to the person skilled in the art,such as, for instance, by an Alphascreen assay or by a FACS competitionassay (e.g. as described herein). Preferably, the blocking activity isdetermined by a FACS competition assay as described in Example 9.Preferably, the ISV has a blocking activity or competition capacity inCHO cells of blocking or competing HRG1-β1 binding to HER3 with an IC50of less than 600 nM, but preferably, 500 nM, 400 nM, 300 nM, 200 nM, 100nM or even less.

-   -   For instance, the 04C07-like ISV has a blocking activity or        competition capacity in this assay with an IC50 of less than 100        nM, more preferably, less than 75 nM, 50 nM or even less, such        as less than 20 nM or 15 nM, 10 nM, 9 nM, 8 nM, 7 nM or 6 nM or        even more preferably of less than 5 nM.    -   For instance, the 17B05-like ISV has a blocking activity or        competition capacity in this assay with an IC50 of less than 150        nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM.    -   For instance, the 21F06-like ISV has a blocking activity or        competition capacity in this assay with an IC50 of less than 100        nM, more preferably, less than 80 nM, 70 nM or even less, such        as less than 60 nM or 50 nM, 40 nM, 30 nM, 20 nM, 15 nM or 13 nM        or even more preferably of less than 11 nM.        In one specific, but non-limiting aspect, (some of the)        “HRG-blocking amino acid sequences” or “HRG-blocking building        blocks” may (and preferably also are) be such that they are        capable of inhibiting or blocking HER3 signaling (see Examples 9        and 10), for example in the phosphorylation assay used in        Example 10. Preferably, any HRG-blocking amino acid sequences,        HRG-blocking building blocks or HRG-blocking ISV's are such that        they have blocking activity, i.e. block or inhibit HRG mediated        HER3 phosphorylation partially or completely, which can be        determined by any suitable assay known to the person skilled in        the art, such as, for instance, by any suitable phosphorylation        assay, such as, for instance, an HER3 phosphorylation assay, an        AKT phosphorylation assay or ERK1/2 phosphorylation assay as        described herein.        Preferably, the blocking activity or inhibiting capacity of        phosphorylation is determined by a HER3 phosphorylation assay as        described in Example 10. Preferably, the ISV has a blocking        activity or an inhibition capacity of ligand (e.g. HRG1-β1)        induced pHER3 phosphorylation in MCF-7 cells with an IC50 of        less than 600 nM, but preferably, 500 nM, 400 nM, 300 nM, 200        nM, 100 nM or even less.    -   For instance, the 04C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        100 nM, more preferably, less than 75 nM, 50 nM or even less,        such as less than 20 nM or 15 nM, 10 nM, 9 nM, 8 nM, 7 nM or 6        nM or even more preferably of less than 5, 4, 3 or 2 nM.    -   For instance, the 17B05-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        15 nM, 10 nM, 9 nM, 8 nM or 7 nM or even more preferably of less        than 6 nM.    -   For instance, the 21F06-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        15 nM, 10 nM, 9 nM, 8 nM or even more preferably of less than 7        nM.

Preferably, the blocking activity or inhibiting capacity of signaling isdetermined by an AKT phosphorylation assay as described in Example 10.1.Preferably, the ISV has a blocking activity or an inhibition capacity ofligand (e.g. HRG1-β1) induced Akt-phosphorylation in MCF-7 cells with anIC50 of less than 600 nM, but preferably, 500 nM, 400 nM, 300 nM, 200nM, 100 nM or even less.

-   -   For instance, the 04C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        100 nM, more preferably, less than 80 nM, 70 nM or even less,        such as less than 60 nM or 50 nM, 45 nM, 40 nM, 35 nM, 30 nM or        even more preferably of less than 20 nM.    -   For instance, the 17B05-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        15 nM, 12 nM, 11 nM, 10 nM or 9 nM or even more preferably of        less than 8 nM.    -   For instance, the 21F06-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 55 nM or 50 nM or even        more preferably of less than 45 nM, such as less than 40 nM or        35 nM, 30 nM, 27.5 nM, 25 nM or even more preferably of less        than 24 nM.

Preferably, the blocking activity or inhibiting capacity of signaling isdetermined by an ERK1/2 phosphorylation assay as described in Example10.2. Preferably, the ISV has a blocking activity or an inhibitioncapacity of ligand (e.g. HRG1-β1) induced ERK1/2-phosphorylation inMCF-7 cells with an IC50M of less than 600 nM, but preferably, 500 nM,400 nM, 300 nM, 200 nM, 150 nM or even less.

-   -   For instance, the 04C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 80 nM, 70 nM or 60 nM, 55 nM or 50 nM or        even more preferably of less than 45 nM, such as less than 40 nM        or 35 nM or even more preferably of less than 30 nM.    -   For instance, the 17B05-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        15 nM, 10 nM, 7.5 nM, 5 nM or 4 nM or even more preferably of        less than 3 nM.    -   For instance, the 21F06-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, such as preferably less than 120 nM.

In yet another specific but non-limiting aspect, an HRG-blocking aminoacid sequence (or HRG-blocking ISV) is an amino acid sequence (or ISV)that competes with either the amino acid sequence 21F06 (SEQ ID NO: 22)and/or the amino acid sequence 04C07 (SEQ ID NO: 15) for binding to HER3and/or that is capable of cross-blocking (as defined herein) the bindingof 21F06 and/or of 04C07 to HER3, for example and in particular in theassay described in Example 2 (section 2.9). Some preferred, butnon-limiting examples of such HRG-blocking ISV's are the “21F06-likesequences” and the “04C07-like sequences” as further described herein.Some of the 21F06-like sequences may be non-limiting examples of ISV'sof the invention that are capable of both blocking/inhibiting ligandbinding as well as inhibiting/blocking signaling. Similarly, the17B05-like sequences described herein are examples of ISV's of theinvention that are capable of both blocking/inhibiting(trans)phosphorylation as well as ligand/HRG binding.

The invention provides amino acid sequences that are directed against(as defined herein) HER3 and that are capable of inhibiting or blocking(fully or partially, as further described herein) (hetero)dimerisationof HER3 (as further described herein), such as EGFR/HER1-HER3(hetero)dimerisation (see for example Examples 13 and 16) and/orHER-2/HER3 (hetero)dimersation), for example in the HER-1/HER3(hetero)dimerisation assay used in Example 13. These amino acidsequences are also referred to herein as “dimerisation-blocking aminoacid sequences” or “dimerisation-blocking building blocks”. Preferably,these dimerisation-blocking amino acid sequences are ISV's (as describedherein), in which case they are also referred to as“dimerisation-blocking ISV's”. Preferably, any dimerisation-blockingamino acid sequences, dimerisation-blocking building blocks ordimerisation-blocking ISV's are such that they block or inhibit(hetero)dimerisation, i.e. block or inhibit dimerisation of HER3 withMET, EGFR and/or HER2 partially or completely, which can be determinedby any suitable assay known to the person skilled in the art, such as,for instance, by a transphosphorylation assay (e.g. as describedherein). Preferably, the blocking or inhibiting capacity is determinedby a transphosphorylation assay as described in Example 10 or 13, forinstance, by determining the EGFR ligand (e.g. TGF-α) induced HER3transphosphorylation as measured in cellular assay in MDA MB468 cells orCHO EGFR/HER3 cells.

-   -   Preferably, the ISV has a blocking or inhibiting activity of        (hetero)dimerisation in CHO EGFR/HER3 cells of blocking or        inhibiting dimerisation of HER3 with EGFR with an IC50 of less        than 600 nM, but preferably, 500 nM, 400 nM, 300 nM, 200 nM, 100        nM or even less.    -   For instance, the 17B05-like ISV has a blocking activity or        inhibiting capacity of in this assay with an IC50 of less than        100 nM, more preferably, less than 75 nM, 50 nM or even less,        such as less than 20 nM or 15 nM, 10 nM, 5 nM, 4 nM, 3 nM or 2        nM or even more preferably of less than 1 nM.

In one specific, but non-limiting aspect, (some of the)dimerisation-blocking amino acid sequences or dimerisation-blockingbuilding blocks may (and preferably also are) such that they are capableof inhibiting or blocking (fully or partially, as further describedherein) ligand binding, and in particular of inhibiting or blocking(fully or partially, as further described herein) the binding of HRG toHER3. In one specific but non-limiting aspect, a dimerisation-blockingamino acid sequence (or dimerisation-blocking ISV) is an amino acidsequence (or ISV) that competes with the amino acid sequence 17B05 (SEQID NO: 13) for binding to HER3 and/or that is capable of cross-blocking(as defined herein) the binding of 17B05 to HER3, for example and inparticular in the assay described in Example 2 (section 2.9) and Example8. Some preferred, but non-limiting examples of suchdimerisation-blocking ISV's are the “17B05-like sequences” as furtherdescribed herein. At least some of these 17B05-like sequences are alsonon-limiting examples of ISV's of the invention that are capable of bothblocking/inhibiting (trans)phosphorylation as well as ligand/HRGbinding. The invention provides amino acid sequences that are directedagainst (as defined herein) HER3 and that are capable of binding todomain II of HER3. These amino acid sequences are also referred toherein as “domain II-binding amino acid sequences” or “domain II-bindingbuilding blocks”. Preferably, these domain II-binding amino acidsequences are ISV's (as described herein), in which case they are alsoreferred to as “domain II-binding ISV's”. Preferably, any domainII-binding amino acid sequences, domain II-binding building blocks ordomain II-binding ISV's are such that they bind to domain II of HER3,which can be determined by any suitable assay known to the personskilled in the art, such as, for instance, by epitope competition ordomain swapping assays (e.g. as described herein). Preferably, thebinding capacity to domain II is determined by binding to chimeric HER3proteins as described in Example 8, for instance, by swapping human HER3domains with chicken HER3 domains.

The domain II-binding amino acid sequences/ISV's or domain II-bindingbuilding blocks/ISV's provided by the invention may also have an effect(which may be limited/partial or more pronounced) on either ligand/HRGbinding (in particular, they may to a limited/partial extent be capableof inhibiting or blocking the binding of ligand/HRG to HER3) and/or onthe (hetero)dimerization of HER3.

In one specific, but non-limiting aspect, (some of the) “domainII-binding amino acid sequences” or “domain II-binding building blocks”may (and preferably also are) be such that they are capable ofinhibiting or blocking HER3 phosphorylation (see Example 10), forinstance in the phosphorylation assay used in Example 10. Preferably,any domain II-binding amino acid sequences or domain II-binding buildingblocks or domain II-binding ISV's are such that they have blockingactivity, i.e. block or inhibit HRG mediated HER3 phosphorylationpartially or completely, which can be determined by any suitable assayknown to the person skilled in the art, such as, for instance, by anysuitable phosphorylation assay, such as, for instance, an HER3phosphorylation assay, an AKT phosphorylation assay or ERK1/2phosphorylation assay as described herein.

Preferably, the blocking activity or inhibiting capacity ofphosphorylation is determined by a HER3 phosphorylation assay asdescribed in Example 10. Preferably, the ISV has a blocking activity oran inhibition capacity of ligand (e.g. HRG1-β1) induced pHER3phosphorylation in MCF-7 cells with an IC50 of less than 600 nM, butpreferably, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM or even less.

-   -   For instance, the 18G11-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        15 nM, 14 nM, 13 nM, 12 nM or even more preferably of less than        11 nM.    -   For instance, the 34C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM, 90 nM, 80 nM or even        less, such as less than 70 nM or 60 nM, 50 nM or 40 nM or even        more preferably of less than 35 nM, such as less than 20 nM or        16 nM, 15 nM, 14 nM, 13 nM or even more preferably of less than        12 nM.

Preferably, the blocking activity or inhibiting capacity ofphosphorylation is determined by an AKT phosphorylation assay asdescribed in Example 10.1. Preferably, the ISV has a blocking activityor an inhibition capacity of ligand (e.g. HRG1-β1) inducedAkt-phosphorylation in MCF-7 cells with an IC50 of less than 600 nM, butpreferably, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM or even less.

-   -   For instance, the 18G11-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 140 nM.    -   For instance, the 34C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, more preferably, less than 100 nM or 90 nM, 80 nM, 70        nM, 60 nM or even more preferably of less than 50 nM.

Preferably, the blocking activity or inhibiting capacity ofphosphorylation is determined by an ERK1/2 phosphorylation assay asdescribed in Example 10.2. Preferably, the ISV has a blocking activityor an inhibition capacity of ligand (e.g. HRG1-β1) inducedERK1/2-phosphorylation in MCF-7 cells with an IC50M of less than 600 nM,but preferably, 500 nM, 400 nM, 300 nM, 200 nM, 150 nM or even less.

-   -   For instance, the 34C07-like ISV has a blocking activity or        competition capacity of in this assay with an IC50 of less than        150 nM, such as less than 120 nM.

In one specific but non-limiting aspect, a domain II-binding amino acidsequence (or domain II-binding ISV) is an amino acid sequence (or ISV)that competes with the amino acid sequence 18G11 (SEQ ID NO: 16) and/orwith the amino acid sequence 34C07 (SEQ ID NO: 18) for binding to HER3and/or that is capable of cross-blocking (as defined herein) the bindingof 18G11 and/or of 34C07 to HER3, for example and in particular in theassay described in Example 2 (section 2.9). Also, in one specific butnon-limiting aspect, a domain II-binding amino acid sequence is capableof inhibiting or blocking HER3 phosphorylation (see Examples 9 and 10),for example in the phosphorylation assay used in Example 10, preferablyessentially without blocking or substantially inhibiting ligand binding.Some preferred, but non-limiting examples of such domain II-bindingISV's are the “18G11-like sequences” and the “34C07-like sequences” asfurther described herein. Of these, some of the 34C07-like sequences areexamples of ISV's of the invention that not only bind to domain II, butalso to a limited/partial extent are capable of inhibiting ligand/HER3binding.

Also, in the present description and claims, the following terms aredefined as follows:

-   A) 21F06-like sequences: a “21F06-like sequence”,“21F06-like ISV” or    “21F06-like building block” is defined as an ISV (as described    herein) that comprises:    -   a) a CDR1 which comprises or essentially consists of either (i)        the amino acid sequence LNAMG (SEQ ID NO: 67) or (ii) an amino        acid sequence that has only 3, 2 or 1 amino acid difference(s)        (as defined herein) with the amino acid sequence LNAMG; and/or    -   b) a CDR2 which comprises or essentially consists of either (i)        the amino acid sequence AIDWSDGNKDYADSVKG (SEQ ID NO: 97)        or (ii) an amino acid sequence that has at least 80%, such as at        least 85%, for example at least 90% or more than 95% sequence        identity with the amino acid sequence AIDWSDGNKDYADSVKG;        or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2        or 1 amino acid difference(s) (as defined herein) with the amino        acid sequence AIDWSDGNKDYADSVKG; and/or    -   c) a CDR3 which comprises or essentially consists of either (i)        the amino acid sequence DTPPWGPMIYIESYDS (SEQ ID NO: 127)        or (ii) an amino acid sequence that has at least 80%, such as at        least 85%, for example at least 90% or more than 95% sequence        identity with the amino acid sequence DTPPWGPMIYIESYDS; or (iii)        an amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence DTPPWGPMIYIESYDS;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 21F06-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

As also mentioned herein, (some of the) 21F06-like sequences may (andpreferably also are) be such that they are capable of inhibiting orblocking HER3 phosphorylation (see Examples 9 and 10), for example inthe phosphorylation assay used in Example 10. Preferably, in such a21F06-like sequence, CDR1 and CDR2 are as defined under a) and b),respectively; or CDR1 and CDR3 are as defined under a) and c),respectively; or CDR2 and CDR3 are as defined under b) and c),respectively. More preferably, in such a 21F06-like sequence, CDR1, CDR2and CDR3 are all as defined under a), b) and c), respectively. Again, insuch an 21F06-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 21F06-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above above.

For example, in such an 21F06-like sequence: CDR1 may comprise oressentially consist of the amino acid sequence LNAMG (with CDR2 and CDR3being as defined under b) and c), respectively); and/or CDR2 maycomprise or essentially consist of the amino acid sequenceAIDWSDGNKDYADSVKG (with CDR1 and CDR3 being as defined under a) and c),respectively); and/or CDR3 may comprise or essentially consist of theamino acid sequence DTPPWGPMIYIESYDS (with CDR1 and CDR2 being asdefined under a) and b), respectively). Particularly, when an 21F06-likesequence is according to this aspect: CDR1 may comprise or essentiallyconsist of the amino acid sequence LNAMG and CDR2 may comprise oressentially consist of the amino acid sequence AIDWSDGNKDYADSVKG (withCDR3 being as defined under c) above); and/or CDR1 may comprise oressentially consist of the amino acid sequence LNAMG and CDR3 maycomprise or essentially consist of the amino acid sequenceDTPPWGPMIYIESYDS (with CDR2 being as defined under b) above); and/orCDR2 may comprise or essentially consist of the amino acid sequenceAIDWSDGNKDYADSVKG and CDR3 may comprise or essentially consist of theamino acid sequence DTPPWGPMIYIESYDS (with CDR1 being as defined undera) above). Again, in such 21F06-like sequences, CDR1, CDR2 and CDR3 arepreferably such that the 21F06-like ISV has blocking activity, e.g.block HRG binding to HER3 partially or completely as described above,and/or blocking activity or inhibiting capacity of phosphorylation in aHER3 phosphorylation assay, and/or pAKT phosphorylation assay, and/orERK1/2 phosphorylation assay, all as described above. In a specificallypreferred aspect, a “21F06-like sequence”,“21F06-like ISV” or“21F06-like building block” is an ISV that comprises:

-   -   d) a CDR1 which is either (i) the amino acid sequence LNAMG        or (ii) an amino acid sequence that has only 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence LNAMG; and/or    -   e) a CDR2 which is either (i) the amino acid sequence        AIDWSDGNKDYADSVKG or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        AIDWSDGNKDYADSVKG; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence AIDWSDGNKDYADSVKG; and/or    -   f) a CDR3 which is either (i) the amino acid sequence        DTPPWGPMIYIESYDS or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        DTPPWGPMIYIESYDS; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence DTPPWGPMIYIESYDS;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 21F06-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above. Preferably, in a21F06-like sequence according to this specifically preferred aspect,CDR1 and CDR2 are as defined under d) and e), respectively; or CDR1 andCDR3 are as defined under d) and f), respectively; or CDR2 and CDR3 areas defined under e) and f), respectively. More preferably, in such a21F06-like sequence, CDR1, CDR2 and CDR3 are all as defined under d), e)and f), respectively. Again, in such an 21F06-like sequence, CDR1, CDR2and CDR3 are preferably such that the 21F06-like ISV has blockingactivity, e.g. block HRG binding to HER3 partially or completely asdescribed above, and/or blocking activity or inhibiting capacity ofphosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above above.

For example, in a 21F06-like sequence according to this specificallypreferred aspect: CDR1 is the amino acid sequence LNAMG (with CDR2 andCDR3 being as defined under e) and f), respectively); and/or CDR2 is theamino acid sequence AIDWSDGNKDYADSVKG (with CDR1 and CDR3 being asdefined under d) and f), respectively); and/or CDR3 is the amino acidsequence DTPPWGPMIYIESYDS (with CDR1 and CDR2 being as defined under d)and e), respectively). Particularly, when an 21F06-like sequence isaccording to this aspect: CDR1 is the amino acid sequence LNAMG and CDR2is the amino acid sequence AIDWSDGNKDYADSVKG (with CDR3 being as definedunder 0 above); and/or CDR1 is the amino acid sequence LNAMG and CDR3 isthe amino acid sequence DTPPWGPMIYIESYDS (with CDR2 being as definedunder e) above); and/or CDR2 is the amino acid sequenceAIDWSDGNKDYADSVKG and CDR3 is DTPPWGPMIYIESYDS (with CDR1 being asdefined under d) above). Again, in such 21F06-like sequences, CDR1, CDR2and CDR3 are preferably such that the 21F06-like ISV has blockingactivity, e.g. block HRG binding to HER3 partially or completely asdescribed above, and/or blocking activity or inhibiting capacity ofphosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above above.

In a particularly preferred 21F06-like sequence: CDR1 is the amino acidsequence LNAMG, CDR2 is the amino acid sequence AIDWSDGNKDYADSVKG; andCDR3 is the amino acid sequence DTPPWGPMIYIESYDS.

In all the 21F06-like sequence described in this paragraph A), theframework sequences may be as further described herein. Preferably, theframework sequences are such that the framework sequences have at least80%, such as at least 85%, for example at least 90%, such as at least95% sequence identity with the framework sequences of 21F06 (which, forexample, can be determined by determining the overall degree of sequenceidentity of a given sequence with the sequence of 21F06 whiledisregarding the CDR's in the calculation). Again, the combination ofCDR's and frameworks present in a given sequence are preferably suchthat the resulting 21F06-like ISV has blocking activity, e.g. block HRGbinding to HER3 partially or completely as described above, and/orblocking activity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above. In one specific aspect, a21F06-like sequence is an ISV that has at least 70%, such at least 80%,for example at least 85%, such as at least 90% or more than 95% sequenceidentity with the amino acid sequence 21F06 (SEQ ID NO: 22). Forexample, in an 21F06-like sequence according to this aspect, the CDR'smay be according to the specifically preferred aspect described above,and may in particularly (but without limitation) be LNAMG (CDR1);AIDWSDGNKDYADSVKG (CDR2); and DTPPWGPMIYIESYDS (CDR3). Again,preferably, the combination of CDR's and frameworks present in such a21F06-like ISV are preferably such that the resulting 21F06-like ISV has

blocking activity, e.g. block HRG binding to HER3 partially orcompletely as described above, and/or blocking activity or inhibitingcapacity of phosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above.

In one particular aspect, any 21F06-like sequence may be a humanizedand/or sequence optimized sequence, as further described herein.

-   B) 04C07-like sequences: a “04C07-like sequence”, “04C07-like ISV”    or “04C07-like building block” is defined as an ISV (as described    herein) that comprises:    -   a) a CDR1 which comprises or essentially consists of either (i)        the amino acid sequence SYPMS (SEQ ID NO: 60) or (ii) an amino        acid sequence that has only 3, 2 or 1 amino acid difference(s)        (as defined herein) with the amino acid sequence SYPMS; and/or    -   b) a CDR2 which comprises or essentially consists of either (i)        the amino acid sequence TVSPGGITTSYADSVKG (SEQ ID NO: 90)        or (ii) an amino acid sequence that has at least 80%, such as at        least 85%, for example at least 90% or more than 95% sequence        identity with the amino acid sequence TVSPGGITTSYADSVKG;        or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2        or 1 amino acid difference(s) (as defined herein) with the amino        acid sequence TVSPGGITTSYADSVKG; and/or    -   c) a CDR3 which comprises or essentially consists of either (i)        the amino acid sequence DLNN (SEQ ID NO: 120) or (ii) an amino        acid sequence that has at least 50%, such as at least 75%        sequence identity with the amino acid sequence DLNN; or (iii) an        amino acid sequence that has only 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        DLNN;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 04C07-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

Preferably, in such a 04C07-like sequence, CDR1 and CDR2 are as definedunder a) and b), respectively; or CDR1 and CDR3 are as defined under a)and c), respectively; or CDR2 and CDR3 are as defined under b) and c),respectively. More preferably, in such a 04C07-like sequence, CDR1, CDR2and CDR3 are all as defined under a), b) and c), respectively. Again, insuch an 04C07-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 04C07-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above above.

For example, in such an 04C07-like sequence: CDR1 may comprise oressentially consist of the amino acid sequence SYPMS (with CDR2 and CDR3being as defined under b) and c), respectively); and/or CDR2 maycomprise or essentially consist of the amino acid sequenceTVSPGGITTSYADSVKG (with CDR1 and CDR3 being as defined under a) and c),respectively); and/or CDR3 may comprise or essentially consist of theamino acid sequence DLNN (with CDR1 and CDR2 being as defined under a)and b), respectively). Particularly, when an 04C07-like sequence isaccording to this aspect: CDR1 may comprise or essentially consist ofthe amino acid sequence SYPMS and CDR2 may comprise or essentiallyconsist of the amino acid sequence TVSPGGITTSYADSVKG (with CDR3 being asdefined under c) above); and/or CDR1 may comprise or essentially consistof the amino acid sequence SYPMS and CDR3 may comprise or essentiallyconsist of the amino acid sequence DLNN (with CDR2 being as definedunder b) above); and/or CDR2 may comprise or essentially consist of theamino acid sequence TVSPGGITTSYADSVKG and CDR3 may comprise oressentially consist of the amino acid sequence DLNN (with CDR1 being asdefined under a) above). Again, in such 04C07-like sequences, CDR1, CDR2and CDR3 are preferably such that the 04C07-like ISV has blockingactivity, e.g. block HRG binding to HER3 partially or completely asdescribed above, and/or blocking activity or inhibiting capacity ofphosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above.

In a specifically preferred aspect, a “04C07-like sequence”, “04C07-likeISV” or “04C07-like building block” is an ISV that comprises:

-   -   d) a CDR1 which is either (i) the amino acid sequence SYPMS        or (ii) an amino acid sequence that has only 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence SYPMS; and/or    -   e) a CDR2 which is either (i) the amino acid sequence        TVSPGGITTSYADSVKG or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        TVSPGGITTSYADSVKG; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence TVSPGGITTSYADSVKG; and/or    -   f) a CDR3 which is either (i) the amino acid sequence DLNN        or (ii) an amino acid sequence that has at least 50%, such as at        least 75%, sequence identity with the amino acid sequence DLNN;        or (iii) an amino acid sequence that has only 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        DLNN;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 04C07-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

Preferably, in a 04C07-like sequence according to this specificallypreferred aspect, CDR1 and CDR2 are as defined under d) and e),respectively; or CDR1 and CDR3 are as defined under d) and f),respectively; or CDR2 and CDR3 are as defined under e) and f),respectively. More preferably, in such a 04C07-like sequence, CDR1, CDR2and CDR3 are all as defined under d), e) and f), respectively. Again, insuch an 04C07-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 04C07-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

For example, in a 04C07-like sequence according to this specificallypreferred aspect: CDR1 is the amino acid sequence SYPMS (with CDR2 andCDR3 being as defined under e) and f), respectively); and/or CDR2 is theamino acid sequence TVSPGGITTSYADSVKG (with CDR1 and CDR3 being asdefined under d) and f), respectively); and/or CDR3 is the amino acidsequence DLNN (with CDR1 and CDR2 being as defined under d) and e),respectively). Particularly, when an 04C07-like sequence is according tothis aspect: CDR1 is the amino acid sequence SYPMS and CDR2 is the aminoacid sequence TVSPGGITTSYADSVKG (with CDR3 being as defined under f)above); and/or CDR1 is the amino acid sequence SYPMS and CDR3 is theamino acid sequence DLNN (with CDR2 being as defined under e) above);and/or CDR2 is the amino acid sequence TVSPGGITTSYADSVKG and CDR3 isDLNN (with CDR1 being as defined under d) above). Again, in such04C07-like sequences, CDR1, CDR2 and CDR3 are preferably such that the04C07-like ISV has blocking activity, e.g. block HRG binding to HER3partially or completely as described above, and/or blocking activity orinhibiting capacity of phosphorylation in a HER3 phosphorylation assay,and/or pAKT phosphorylation assay, and/or ERK1/2 phosphorylation assay,all as described above.

In a particularly preferred 04C07-like sequence: CDR1 is the amino acidsequence SYPMS, CDR2 is the amino acid sequence TVSPGGITTSYADSVKG; andCDR3 is the amino acid sequence DLNN.

In all the 04C07-like sequence described in this paragraph B), theframework sequences may be as further described herein. Preferably, theframework sequences are such that the framework sequences have at least80%, such as at least 85%, for example at least 90%, such as at least95% sequence identity with the framework sequences of 04C07 (which, forexample, can be determined by determining the overall degree of sequenceidentity of a given sequence with the sequence of 04C07 whiledisregarding the CDR's in the calculation). Again, the combination ofCDR's and frameworks present in a given sequence are preferably suchthat the resulting 04C07-like ISV has blocking activity, e.g. block HRGbinding to HER3 partially or completely as described above, and/orblocking activity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

In one specific aspect, a 04C07-like sequence is an ISV that has atleast 70%, such at least 80%, for example at least 85%, such as at least90% or more than 95% sequence identity with the amino acid sequence04C07 (SEQ ID NO: 15). For example, in an 04C07-like sequence accordingto this aspect, the CDR's may be according to the specifically preferredaspect described above, and may in particularly (but without limitation)be SYPMS (CDR1); TVSPGGITTSYADSVKG (CDR2); and DLNN (CDR3). Again,preferably, the combination of CDR's and frameworks present in such a04C07-like ISV are preferably such that the resulting 04C07-like ISV hasblocking activity, e.g. block HRG binding to HER3 partially orcompletely as described above, and/or blocking activity or inhibitingcapacity of phosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above.

In one particular aspect, any 04C07-like sequence may be a humanizedand/or sequence optimized sequence, as further described herein.

-   C) 17B05-like sequences: a “17B05-like sequence”, “17B05-like ISV”    or “17B05-like building block” is defined as an ISV (as described    herein) that comprises:    -   a) a CDR1 which comprises or essentially consists of either (i)        the amino acid sequence LNAMA (SEQ ID NO: 58) or (ii) an amino        acid sequence that has only 3, 2 or 1 amino acid difference(s)        (as defined herein) with the amino acid sequence LNAMA; and/or    -   b) a CDR2 which comprises or essentially consists of either (i)        the amino acid sequence GIFGVGSTRYADSVKG (SEQ ID NO: 88) or (ii)        an amino acid sequence that has at least 80%, such as at least        85%, for example at least 90% or more than 95% sequence identity        with the amino acid sequence GIFGVGSTRYADSVKG; or (iii) an amino        acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        GIFGVGSTRYADSVKG; and/or    -   c) a CDR3 which comprises or essentially consists of either (i)        the amino acid sequence SSVTRGSSDY (SEQ ID NO: 118) or (ii) an        amino acid sequence that has at least 80%, such as at least 85%,        for example at least 90% or more than 95% sequence identity with        the amino acid sequence SSVTRGSSDY; or (iii) an amino acid        sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        SSVTRGSSDY;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 17B05-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of (trans)phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

As mentioned herein, (some of the) 17B05-like sequences may be (andpreferably are) such that they are capable of inhibiting or blocking(fully or partially, as further described herein) ligand binding, and inparticular of inhibiting or blocking (fully or partially, as furtherdescribed herein) the binding of HRG to HER3

Preferably, in such a 17B05-like sequence, CDR1 and CDR2 are as definedunder a) and b), respectively; or CDR1 and CDR3 are as defined under a)and c), respectively; or CDR2 and CDR3 are as defined under b) and c),respectively. More preferably, in such a 17B05-like sequence, CDR1, CDR2and CDR3 are all as defined under a), b) and c), respectively. Again, insuch an 17B05-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 17B05-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of (trans)phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

For example, in such an 17B05-like sequence: CDR1 may comprise oressentially consist of the amino acid sequence LNAMA (with CDR2 and CDR3being as defined under b) and c), respectively); and/or CDR2 maycomprise or essentially consist of the amino acid sequenceGIFGVGSTRYADSVKG (with CDR1 and CDR3 being as defined under a) and c),respectively); and/or CDR3 may comprise or essentially consist of theamino acid sequence SSVTRGSSDY (with CDR1 and CDR2 being as definedunder a) and b), respectively). Particularly, when an 17B05-likesequence is according to this aspect: CDR1 may comprise or essentiallyconsist of the amino acid sequence LNAMA and CDR2 may comprise oressentially consist of the amino acid sequence GIFGVGSTRYADSVKG (withCDR3 being as defined under c) above); and/or CDR1 may comprise oressentially consist of the amino acid sequence LNAMA and CDR3 maycomprise or essentially consist of the amino acid sequence SSVTRGSSDY(with CDR2 being as defined under b) above); and/or CDR2 may comprise oressentially consist of the amino acid sequence GIFGVGSTRYADSVKG and CDR3may comprise or essentially consist of the amino acid sequenceSSVTRGSSDY (with CDR1 being as defined under a) above). Again, in such17B05-like sequences, CDR1, CDR2 and CDR3 are preferably such that the17B05-like ISV has blocking activity, e.g. block HRG binding to HER3partially or completely as described above, and/or blocking activity orinhibiting capacity of (trans)phosphorylation in a HER3 phosphorylationassay, and/or pAKT phosphorylation assay, and/or ERK1/2 phosphorylationassay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

In a specifically preferred aspect, a “17B05-like sequence”, “17B05-likeISV” or “/7B05-like building block” is an ISV that comprises:

-   -   d) a CDR1 which is either (i) the amino acid sequence LNAMA        or (ii) an amino acid sequence that has only 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence LNAMA; and/or    -   e) a CDR2 which is either (i) the amino acid sequence        GIFGVGSTRYADSVKG or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        GIFGVGSTRYADSVKG; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence GIFGVGSTRYADSVKG; and/or    -   f) a CDR3 which is either (i) the amino acid sequence SSVTRGSSDY        or (ii) an amino acid sequence that has at least 80%, such as at        least 85%, for example at least 90% or more than 95% sequence        identity with the amino acid sequence SSVTRGSSDY; or (iii) an        amino acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence SSVTRGSSDY;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 17B05-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of (trans)phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

Preferably, in a 17B05-like sequence according to this specificallypreferred aspect, CDR1 and CDR2 are as defined under d) and e),respectively; or CDR1 and CDR3 are as defined under d) and f),respectively; or CDR2 and CDR3 are as defined under e) and f),respectively. More preferably, in such a 17B05-like sequence, CDR1, CDR2and CDR3 are all as defined under d), e) and f), respectively. Again, insuch an 17B05-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 17B05-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of (trans)phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

For example, in a 17B05-like sequence according to this specificallypreferred aspect: CDR1 is the amino acid sequence LNAMA (with CDR2 andCDR3 being as defined under e) and f), respectively); and/or CDR2 is theamino acid sequence GIFGVGSTRYADSVKG (with CDR1 and CDR3 being asdefined under d) and f), respectively); and/or CDR3 is the amino acidsequence SSVTRGSSDY (with CDR1 and CDR2 being as defined under d) ande), respectively). Particularly, when an 17B05-like sequence isaccording to this aspect: CDR1 is the amino acid sequence LNAMA and CDR2is the amino acid sequence GIFGVGSTRYADSVKG (with CDR3 being as definedunder f) above); and/or CDR1 is the amino acid sequence LNAMA and CDR3is the amino acid sequence SSVTRGSSDY (with CDR2 being as defined undere) above); and/or CDR2 is the amino acid sequence GIFGVGSTRYADSVKG andCDR3 is SSVTRGSSDY (with CDR1 being as defined under d) above). Again,in such 17B05-like sequences, CDR1, CDR2 and CDR3 are preferably suchthat the 17B05-like ISV has blocking activity, e.g. block HRG binding toHER3 partially or completely as described above, and/or blockingactivity or inhibiting capacity of (trans)phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay and/or has a blocking or inhibiting activity of(hetero)dimerisation as determined by EGFR ligand (EGF) induced HER3phosphorylation assay, all as described above.

In a particularly preferred 17B05-like sequence: CDR1 is the amino acidsequence LNAMA, CDR2 is the amino acid sequence GIFGVGSTRYADSVKG; andCDR3 is the amino acid sequence SSVTRGSSDY.

In all the 17B05-like sequence described in this paragraph C), theframework sequences may be as further described herein. Preferably, theframework sequences are such that the framework sequences have at least80%, such as at least 85%, for example at least 90%, such as at least95% sequence identity with the framework sequences of 17B05 (which, forexample, can be determined by determining the overall degree of sequenceidentity of a given sequence with the sequence of 17B05 whiledisregarding the CDR's in the calculation). Again, the combination ofCDR's and frameworks present in a given sequence are preferably suchthat the resulting 17B05-like ISV has blocking activity, e.g. block HRGbinding to HER3 partially or completely as described above, and/orblocking activity or inhibiting capacity of (trans)phosphorylation in aHER3 phosphorylation assay, and/or pAKT phosphorylation assay, and/orERK1/2 phosphorylation assay and/or has a blocking or inhibitingactivity of (hetero)dimerisation as determined by EGFR ligand (EGF)induced HER3 phosphorylation assay, all as described above.

In one specific aspect, a 17B05-like sequence is an ISV that has atleast 70%, such at least 80%, for example at least 85%, such as at least90% or more than 95% sequence identity with the amino acid sequence17B05 (SEQ ID NO: 13). For example, in an 17B05-like sequence accordingto this aspect, the CDR's may be according to the specifically preferredaspect described above, and may in particularly (but without limitation)be LNAMA (CDR1); GIFGVGSTRYADSVKG (CDR2); and SSVTRGSSDY (CDR3). Again,preferably, the combination of CDR's and frameworks present in such a17B05-like ISV are preferably such that the resulting 17B05-like ISV hasblocking activity, e.g. block HRG binding to HER3 partially orcompletely as described above, and/or blocking activity or inhibitingcapacity of (trans)phosphorylation in a HER3 phosphorylation assay,and/or pAKT phosphorylation assay, and/or ERK1/2 phosphorylation assayand/or has a blocking or inhibiting activity of (hetero)dimerisation asdetermined by EGFR ligand (EGF) induced HER3 phosphorylation assay, allas described above. In one particular aspect, any 17B05-like sequencemay be a humanized and/or sequence optimized sequence, as furtherdescribed herein.

-   D) 18G11-like sequences: a “18G11-like sequence”, “18G11-like ISV”    or “18G11-like building block” is defined as an ISV (as described    herein) that comprises:    -   a) a CDR1 which comprises or essentially consists of either (i)        the amino acid sequence INAMG (SEQ ID NO: 61) or (ii) an amino        acid sequence that has only 3, 2 or 1 amino acid difference(s)        (as defined herein) with the amino acid sequence INAMG; and/or    -   b) a CDR2 which comprises or essentially consists of either (i)        the amino acid sequence LITSSDTTDYAESVEG (SEQ ID NO: 91) or (ii)        an amino acid sequence that has at least 80%, such as at least        85%, for example at least 90% or more than 95% sequence identity        with the amino acid sequence LITSSDTTDYAESVEG; or (iii) an amino        acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        LITSSDTTDYAESVEG; and/or    -   c) a CDR3 which comprises or essentially consists of either (i)        the amino acid sequence DHYSMGVPEKRVIM (SEQ ID NO: 121) or (ii)        an amino acid sequence that has at least 80%, such as at least        85%, for example at least 90% or more than 95% sequence identity        with the amino acid sequence DHYSMGVPEKRVIM; or (iii) an amino        acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        DHYSMGVPEKRVIM;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 18G11-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above above.

As mentioned herein, (some of the) 18G11-like sequences may have aneffect (which may be limited/partial or more pronounced) on eitherligand/HRG binding (in particular, they may to a limited/partial extentbe capable of inhibiting or blocking the binding of ligand/HRG to HER3)and/or on the (hetero)dimerization of HER3.

Preferably, in such a 18G11-like sequence, CDR1 and CDR2 are as definedunder a) and b), respectively; or CDR1 and CDR3 are as defined under a)and c), respectively; or CDR2 and CDR3 are as defined under b) and c),respectively. More preferably, in such a 18G11-like sequence, CDR1, CDR2and CDR3 are all as defined under a), b) and c), respectively. Again, insuch an 18G11-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 18G11-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above.

For example, in such an 18G11-like sequence: CDR1 may comprise oressentially consist of the amino acid sequence INAMG (with CDR2 and CDR3being as defined under b) and c), respectively); and/or CDR2 maycomprise or essentially consist of the amino acid sequenceLITSSDTTDYAESVEG (with CDR1 and CDR3 being as defined under a) and c),respectively); and/or CDR3 may comprise or essentially consist of theamino acid sequence DHYSMGVPEKRVIM (with CDR1 and CDR2 being as definedunder a) and b), respectively). Particularly, when an 18G11-likesequence is according to this aspect: CDR1 may comprise or essentiallyconsist of the amino acid sequence INAMG and CDR2 may comprise oressentially consist of the amino acid sequence LITSSDTTDYAESVEG (withCDR3 being as defined under c) above); and/or CDR1 may comprise oressentially consist of the amino acid sequence INAMG and CDR3 maycomprise or essentially consist of the amino acid sequenceDHYSMGVPEKRVIM (with CDR2 being as defined under b) above); and/or CDR2may comprise or essentially consist of the amino acid sequenceLITSSDTTDYAESVEG and CDR3 may comprise or essentially consist of theamino acid sequence DHYSMGVPEKRVIM (with CDR1 being as defined under a)above). Again, in such 18G11-like sequences, CDR1, CDR2 and CDR3 arepreferably such that the 18G11-like ISV has domain II binding activity,blocking activity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above.

In a specifically preferred aspect, a “18G11-like sequence”, “18G11-likeISV” or “18G11-like building block” is an ISV that comprises:

-   -   d) a CDR1 which is either (i) the amino acid sequence INAMG        or (ii) an amino acid sequence that has only 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence INAMG; and/or    -   e) a CDR2 which is either (i) the amino acid sequence        LITSSDTTDYAESVEG or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        LITSSDTTDYAESVEG; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence LITSSDTTDYAESVEG; and/or    -   f) a CDR3 which is either (i) the amino acid sequence        DHYSMGVPEKRVIM or (ii) an amino acid sequence that has at least        80%, such as at least 85%, for example at least 90% or more than        95% sequence identity with the amino acid sequence        DHYSMGVPEKRVIM; or (iii) an amino acid sequence that has only 7,        6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined herein)        with the amino acid sequence DHYSMGVPEKRVIM;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 18G11-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above above.

Preferably, in a 18G11-like sequence according to this specificallypreferred aspect, CDR1 and CDR2 are as defined under d) and e),respectively; or CDR1 and CDR3 are as defined under d) and f),respectively; or CDR2 and CDR3 are as defined under e) and f),respectively. More preferably, in such a 18G11-like sequence, CDR1, CDR2and CDR3 are all as defined under d), e) and f), respectively. Again, insuch an 18G11-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 18G11-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above. For example, in a 18G11-like sequence according to thisspecifically preferred aspect: CDR1 is the amino acid sequence INAMG(with CDR2 and CDR3 being as defined under e) and f), respectively);and/or CDR2 is the amino acid sequence LITSSDTTDYAESVEG (with CDR1 andCDR3 being as defined under d) and f), respectively); and/or CDR3 is theamino acid sequence DHYSMGVPEKRVIM (with CDR1 and CDR2 being as definedunder d) and e), respectively). Particularly, when an 18G11-likesequence is according to this aspect: CDR1 is the amino acid sequenceINAMG and CDR2 is the amino acid sequence LITSSDTTDYAESVEG (with CDR3being as defined under f) above); and/or CDR1 is the amino acid sequenceINAMG and CDR3 is the amino acid sequence DHYSMGVPEKRVIM (with CDR2being as defined under e) above); and/or CDR2 is the amino acid sequenceLITSSDTTDYAESVEG and CDR3 is DHYSMGVPEKRVIM (with CDR1 being as definedunder d) above). Again, in such 18G11-like sequences, CDR1, CDR2 andCDR3 are preferably such that the 18G11-like ISV has domain II bindingactivity, blocking activity or inhibiting capacity of phosphorylation ina HER3 phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above.

In a particularly preferred 18G11-like sequence: CDR1 is the amino acidsequence INAMG, CDR2 is the amino acid sequence LITSSDTTDYAESVEG; andCDR3 is the amino acid sequence DHYSMGVPEKRVIM.

In all the 18G11-like sequence described in this paragraph D), theframework sequences may be as further described herein. Preferably, theframework sequences are such that the framework sequences have at least80%, such as at least 85%, for example at least 90%, such as at least95% sequence identity with the framework sequences of 18G11 (which, forexample, can be determined by determining the overall degree of sequenceidentity of a given sequence with the sequence of 18G11 whiledisregarding the CDR's in the calculation). Again, the combination ofCDR's and frameworks present in a given sequence are preferably suchthat the resulting 18G11-like ISV has domain II binding activity,blocking activity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay and/or pAKT phosphorylation assay, all asdescribed above. In one specific aspect, a 18G11-like sequence is an ISVthat has at least 70%, such at least 80%, for example at least 85%, suchas at least 90% or more than 95% sequence identity with the amino acidsequence 18G11 (SEQ ID NO: 16). For example, in an 18G11-like sequenceaccording to this aspect, the CDR's may be according to the specificallypreferred aspect described above, and may in particularly (but withoutlimitation) be INAMG (CDR1); LITSSDTTDYAESVEG (CDR2); and DHYSMGVPEKRVIM(CDR3). Again, preferably, the combination of CDR's and frameworkspresent in such a 18G11-like ISV are preferably such that the resulting18G11-like ISV has domain II binding activity, blocking activity orinhibiting capacity of phosphorylation in a HER3 phosphorylation assayand/or pAKT phosphorylation assay, all as described above.

In one particular aspect, any 18G11-like sequence may be a humanizedand/or sequence optimized sequence, as further described herein.

-   E) 34C07-like sequences: a “34C07-like sequence”, “34C07-like ISV”    or “34C07-like building block” is defined as an ISV (as described    herein) that comprises:    -   a) a CDR1 which comprises or essentially consists of either (i)        the amino acid sequence INAMA (SEQ ID NO: 63) or (ii) an amino        acid sequence that has only 3, 2 or 1 amino acid difference(s)        (as defined herein) with the amino acid sequence INAMA; and/or    -   b) a CDR2 which comprises or essentially consists of either (i)        the amino acid sequence EITAGGSTNYADSVKG (SEQ ID NO: 93) or (ii)        an amino acid sequence that has at least 80%, such as at least        85%, for example at least 90% or more than 95% sequence identity        with the amino acid sequence EITAGGSTNYADSVKG; or (iii) an amino        acid sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        EITAGGSTNYADSVKG; and/or    -   c) a CDR3 which comprises or essentially consists of either (i)        the amino acid sequence DHYTTWDRRSAY (SEQ ID NO: 123) or (ii) an        amino acid sequence that has at least 80%, such as at least 85%,        for example at least 90% or more than 95% sequence identity with        the amino acid sequence DHYTTWDRRSAY; or (iii) an amino acid        sequence that has only 7, 6, 5, 4, 3, 2 or 1 amino acid        difference(s) (as defined herein) with the amino acid sequence        DHYTTWDRRSAY;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 34C07-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

As mentioned herein, (some of the) 34C07-like sequences may have aneffect (which may be limited/partial or more pronounced) on eitherligand/HRG binding (in particular, they may to a limited/partial extentbe capable of inhibiting or blocking the binding of ligand/HRG to HER3)and/or on the (hetero)dimerization of HER3. In particular, they may to alimited extent be capable of inhibiting ligand/HRG binding to HER3.Preferably, in such a 34C07-like sequence, CDR1 and CDR2 are as definedunder a) and b), respectively; or CDR1 and CDR3 are as defined under a)and c), respectively; or CDR2 and CDR3 are as defined under b) and c),respectively. More preferably, in such a 34C07-like sequence, CDR1, CDR2and CDR3 are all as defined under a), b) and c), respectively. Again, insuch an 34C07-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 34C07-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above above.

For example, in such an 34C07-like sequence: CDR1 may comprise oressentially consist of the amino acid sequence INAMA (with CDR2 and CDR3being as defined under b) and c), respectively); and/or CDR2 maycomprise or essentially consist of the amino acid sequenceEITAGGSTNYADSVKG (with CDR1 and CDR3 being as defined under a) and c),respectively); and/or CDR3 may comprise or essentially consist of theamino acid sequence DHYTTWDRRSAY (with CDR1 and CDR2 being as definedunder a) and b), respectively). Particularly, when an 34C07-likesequence is according to this aspect: CDR1 may comprise or essentiallyconsist of the amino acid sequence INAMA and CDR2 may comprise oressentially consist of the amino acid sequence EITAGGSTNYADSVKG (withCDR3 being as defined under c) above); and/or CDR1 may comprise oressentially consist of the amino acid sequence INAMA and CDR3 maycomprise or essentially consist of the amino acid sequence DHYTTWDRRSAY(with CDR2 being as defined under b) above); and/or CDR2 may comprise oressentially consist of the amino acid sequence EITAGGSTNYADSVKG and CDR3may comprise or essentially consist of the amino acid sequenceDHYTTWDRRSAY (with CDR1 being as defined under a) above). Again, in such34C07-like sequences, CDR1, CDR2 and CDR3 are preferably such that the34C07-like ISV has domain II binding activity, blocking activity orinhibiting capacity of phosphorylation in a HER3 phosphorylation assay,and/or pAKT phosphorylation assay, and/or ERK1/2 phosphorylation assay,all as described above.

In a specifically preferred aspect, a “34C07-like sequence”, “34C07-likeISV” or “34C07-like building block” is an ISV that comprises:

-   -   d) a CDR1 which is either (i) the amino acid sequence INAMA        or (ii) an amino acid sequence that has only 3, 2 or 1 amino        acid difference(s) (as defined herein) with the amino acid        sequence INAMA; and/or    -   e) a CDR2 which is either (i) the amino acid sequence        EITAGGSTNYADSVKG or (ii) an amino acid sequence that has at        least 80%, such as at least 85%, for example at least 90% or        more than 95% sequence identity with the amino acid sequence        EITAGGSTNYADSVKG; or (iii) an amino acid sequence that has only        7, 6, 5, 4, 3, 2 or 1 amino acid difference(s) (as defined        herein) with the amino acid sequence EITAGGSTNYADSVKG; and/or    -   f) a CDR3 which is either (i) the amino acid sequence        DHYTTWDRRSAY or (ii) an amino acid sequence that has at least        80%, such as at least 85%, for example at least 90% or more than        95% sequence identity with the amino acid sequence DHYTTWDRRSAY;        or (iii) an amino acid sequence that has only 7, 6, 5, 4, 3, 2        or 1 amino acid difference(s) (as defined herein) with the amino        acid sequence DHYTTWDRRSAY;

in which the framework sequences present in such an ISV are as furtherdescribed herein, and in which CDR1, CDR2 and CDR3 are preferably suchthat the 34C07-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

Preferably, in a 34C07-like sequence according to this specificallypreferred aspect, CDR1 and CDR2 are as defined under d) and e),respectively; or CDR1 and CDR3 are as defined under d) and f),respectively; or CDR2 and CDR3 are as defined under e) and f),respectively. More preferably, in such a 34C07-like sequence, CDR1, CDR2and CDR3 are all as defined under d), e) and f), respectively. Again, insuch an 34C07-like sequence, CDR1, CDR2 and CDR3 are preferably suchthat the 34C07-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

For example, in a 34C07-like sequence according to this specificallypreferred aspect: CDR1 is the amino acid sequence INAMA (with CDR2 andCDR3 being as defined under e) and f), respectively); and/or CDR2 is theamino acid sequence EITAGGSTNYADSVKG (with CDR1 and CDR3 being asdefined under d) and f), respectively); and/or CDR3 is the amino acidsequence DHYTTWDRRSAY (with CDR1 and CDR2 being as defined under d) ande), respectively). Particularly, when an 34C07-like sequence isaccording to this aspect: CDR1 is the amino acid sequence INAMA and CDR2is the amino acid sequence EITAGGSTNYADSVKG (with CDR3 being as definedunder f) above); and/or CDR1 is the amino acid sequence INAMA and CDR3is the amino acid sequence DHYTTWDRRSAY (with CDR2 being as definedunder e) above); and/or CDR2 is the amino acid sequence EITAGGSTNYADSVKGand CDR3 is DHYTTWDRRSAY (with CDR1 being as defined under d) above).Again, in such 34C07-like sequences, CDR1, CDR2 and CDR3 are preferablysuch that the 34C07-like ISV has domain II binding activity, blockingactivity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

In a particularly preferred 34C07-like sequence: CDR1 is the amino acidsequence INAMA, CDR2 is the amino acid sequence EITAGGSTNYADSVKG; andCDR3 is the amino acid sequence DHYTTWDRRSAY.

In all the 34C07-like sequence described in this paragraph E), theframework sequences may be as further described herein. Preferably, theframework sequences are such that the framework sequences have at least80%, such as at least 85%, for example at least 90%, such as at least95% sequence identity with the framework sequences of 34C07 (which, forexample, can be determined by determining the overall degree of sequenceidentity of a given sequence with the sequence of 34C07 whiledisregarding the CDR's in the calculation). Again, the combination ofCDR's and frameworks present in a given sequence are preferably suchthat the resulting 34C07-like ISV has domain II binding activity,blocking activity or inhibiting capacity of phosphorylation in a HER3phosphorylation assay, and/or pAKT phosphorylation assay, and/or ERK1/2phosphorylation assay, all as described above.

In one specific aspect, a 34C07-like sequence is an ISV that has atleast 70%, such at least 80%, for example at least 85%, such as at least90% or more than 95% sequence identity with the amino acid sequence34C07 (SEQ ID NO: 18). For example, in an 34C07-like sequence accordingto this aspect, the CDR's may be according to the specifically preferredaspect described above, and may in particularly (but without limitation)be INAMA (CDR1); EITAGGSTNYADSVKG (CDR2); and DHYTTWDRRSAY (CDR3).Again, preferably, the combination of CDR's and frameworks present insuch a 34C07-like ISV are preferably such that the resulting 34C07-likeISV has domain II binding activity, blocking activity or inhibitingcapacity of phosphorylation in a HER3 phosphorylation assay, and/or pAKTphosphorylation assay, and/or ERK1/2 phosphorylation assay, all asdescribed above.

In one particular aspect, any 34C07-like sequence may be a humanizedand/or sequence optimized sequence, as further described herein.

In one particular aspect, any 34C07-like sequence may be a humanizedand/or sequence optimized sequence, as further described herein.

All of the amino acid sequences of the invention as described herein(including those according to specific aspects mentioned herein, such asthe aspects mentioned in the preceding paragraph) that can bind to HER3with an affinity (suitably measured and/or expressed as a K_(D)-value(actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rateand/or a k_(off)-rate, or alternatively as an IC₅₀ value, as furtherdescribed herein) that is as defined herein; as well as compounds andconstructs, and in particular proteins and polypeptides, that compriseat least one such amino acid sequence.

In particular, the amino acid sequences and polypeptides of theinvention are preferably such that they:

-   -   bind to HER3 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER3 with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻, such as between 10⁵        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to HER3 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or apolypeptide that contains only one amino acid sequence of the invention)is such that it will bind to HER3 with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences orpolypeptides of the invention to HER3 will become clear from the furtherdescription and examples herein.

For binding to HER3, an amino acid sequence of the invention willusually contain within its amino acid sequence one or more amino acidresidues or one or more stretches of amino acid residues (i.e. with each“stretch” comprising two or more amino acid residues that are adjacentto each other or in close proximity to each other, i.e. in the primaryor tertiary structure of the amino acid sequence) via which the aminoacid sequence of the invention can bind to HER3, which amino acidresidues or stretches of amino acid residues thus form the “site” forbinding to HER3 (also referred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more amino acid sequences of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than HER3), so as to provide a monovalent,multivalent or multispecific polypeptide of the invention, respectively,all as described herein. Such a protein or polypeptide may also be inessentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as suchpreferably essentially consist of a single amino acid chain that is notlinked via disulphide bridges to any other amino acid sequence or chain(but that may or may not contain one or more intramolecular disulphidebridges. For example, it is known that immunoglobulin single variabledomains and/or Nanobodies—as described herein—may sometimes contain adisulphide bridge between CDR3 and CDR1 or FR2). However, it should benoted that one or more amino acid sequences of the invention may belinked to each other and/or to other amino acid sequences (e.g. viadisulphide bridges) to provide peptide constructs that may also beuseful in the invention (for example Fab′ fragments, F(ab′)₂ fragments,ScFv constructs, “diabodies” and other multispecific constructs.Reference is for example made to the review by Holliger and Hudson, Nat.Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound,construct or polypeptide comprising the same) is intended foradministration to a subject (for example for therapeutic and/ordiagnostic purposes as described herein), it is preferably either anamino acid sequence that does not occur naturally in said subject; or,when it does occur naturally in said subject, in essentially isolatedform (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use,the amino acid sequences of the invention (as well as compounds,constructs and polypeptides comprising the same) are preferably directedagainst human HER3; whereas for veterinary purposes, the amino acidsequences and polypeptides of the invention are preferably directedagainst HER3 from the species to be treated, or at at leastcross-reactive with HER3 from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, andin addition to the at least one binding site for binding against HER3,contain one or more further binding sites for binding against otherantigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of theinvention, and of compositions comprising the same, can be tested usingany suitable in vitro assay, cell-based assay, in vivo assay and/oranimal model known per se, or any combination thereof, depending on thespecific disease or disorder involved. Suitable assays and animal modelswill be clear to the skilled person, and for example include [liganddependent HER3 phosphorylation (Wallasch et. Al. (1995) EMBO J. 14(17):4267-4275), and xenograft tumor models (Schoeberl et. al. (2009) Sci.Signal. 2(77): ra 31)], as well as the assays and animal models used inthe experimental part below and in the prior art cited herein.

Also, according to the invention, amino acid sequences and polypeptidesthat are directed against HER3 from a first species of warm-bloodedanimal may or may not show cross-reactivity with HER3 from one or moreother species of warm-blooded animal. For example, amino acid sequencesand polypeptides directed against human HER3 may or may not show crossreactivity with HER3 from one or more other species of primates (suchas, without limitation, monkeys from the genus Macaca (such as, and inparticular, cynomolgus monkeys (Macaca fascicularis) and/or rhesusmonkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with HER3from one or more species of animals that are often used in animal modelsfor diseases (for example mouse, rat, rabbit, pig or dog), and inparticular in animal models for diseases and disorders associated withHER3 (such as the species and animal models mentioned herein). In thisrespect, it will be clear to the skilled person that suchcross-reactivity, when present, may have advantages from a drugdevelopment point of view, since it allows the amino acid sequences andpolypeptides against human HER3 to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the inventionthat are cross-reactive with HER3 from multiple species of mammal willusually be advantageous for use in veterinary applications, since itwill allow the same amino acid sequence or polypeptide to be used acrossmultiple species. Thus, it is also encompassed within the scope of theinvention that amino acid sequences and polypeptides directed againstHER3 from one species of animal (such as amino acid sequences andpolypeptides against human HER3) can be used in the treatment of anotherspecies of animal, as long as the use of the amino acid sequences and/orpolypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularlylimited to or defined by a specific antigenic determinant, epitope,part, domain, subunit or confirmation (where applicable) of HER3 againstwhich the amino acid sequences and polypeptides of the invention aredirected. For example, the amino acid sequences and polypeptides may ormay not be directed against an “interaction site” (as defined herein).However, it is generally assumed and preferred that the amino acidsequences and polypeptides of the invention are preferably directedagainst an interaction site (as defined herein), and in particularagainst Heregulin binding site and/or heterodimerization site (see Hsiehand Moasser, supra). Thus, as further described herein, in onepreferred, but non-limiting aspect, the amino acid sequences andpolypeptides of the invention are directed against the HER3 ligandbinding site and/or against the heterodimerization site of HER3, and areas further defined herein. It is noted that other HER3 ligands have beendescribed besides Heregulin (Sithanandam and Anderson (2008) Cancer GeneTherapy, supra). Thus, in another preferred, but non limiting aspect,the amino acid sequences and polypeptides of the invention are directedagainst the Heregulin (also referred to herein as “HRG”) binding siteand/or against the heterodimerization site of HER3. As mentioned above,amino acid sequences of the invention that are directed against the HRGbinding site are also referred to herein as “HRG-blocking amino acidsequences”, “HRG-blocking building blocks” or, when they are ISV's,“HRG-blocking ISV's”. Amino acid sequences of the invention that aredirected against the HRG binding site (and that most preferably are alsocapable of inhibiting or blocking HER3 heterodimerization, as furtherdescribed herein) are also referred to herein as “dimerisation-blockingamino acid sequences”,“dimerisation-blocking building blocks” or, whenthey are ISV's, “dimerisation-blocking ISV's”.

As further described herein, a polypeptide of the invention may containtwo or more amino acid sequences of the invention that are directedagainst HER3 and in a preferred aspect contain two different amino acidsequences such as immunoglobulin single variable domains that aredirected against HER3. Generally, such polypeptides will bind to HER3with increased avidity compared to a single amino acid sequence of theinvention. Such a polypeptide may for example comprise two amino acidsequences of the invention that are directed against the same antigenicdeterminant, epitope, part, domain, subunit or confirmation (whereapplicable) of HER3 (which may or may not be an interaction site); orcomprise at least one “first” amino acid sequence of the invention thatis directed against a first same antigenic determinant, epitope, part,domain, subunit or confirmation (where applicable) of HER3 (which maye.g. be the Heregulin interaction site); and at least one “second” aminoacid sequence of the invention that is directed against a secondantigenic determinant, epitope, part, domain, subunit or confirmation(where applicable) different from the first (and which may be e.g. bedirected against the heterodimerization site). Preferably, in such“biparatopic” polypeptides of the invention, at least one amino acidsequence of the invention is directed against an interaction site (asdefined herein), although the invention in its broadest sense is notlimited thereto.

Accordingly, and as further described herein, in one specificallyadvantageous but non-limiting aspect, the invention makes it possible toprovide polypeptides of the invention that are both directed against theHRG binding site and also capable of blocking or inhibiting HER3heterodimerisation, for example by combining one or more (such as one ortwo) HRG-blocking building blocks with one or more (such as one or two)dimerisation-blocking building blocks in a single polypeptide of theinvention (which may be as further described herein).

Also, when the target (i.e. HER3) is part of a binding pair (forexample, a receptor-ligand binding pair), the amino acid sequences andpolypeptides may be such that they compete with the cognate bindingpartner (e.g. the ligand, receptor or other binding partner, asapplicable) for binding to the target, and/or such that they (fully orpartially) neutralize binding of the binding partner to the target. Inthis respect, it should again be noted that, as mentioned above, otherHER3 ligands have been described besides Heregulin (Sithanandam andAnderson (2008) Cancer Gene Therapy, supra), and the amino acidsequences of the invention may (also) compete with and/or (fully orpartially) neutralize binding of such ligands to HER3.

It is also within the scope of the invention that, where applicable, anamino acid sequence of the invention can bind to two or more antigenicdeterminants, epitopes, parts, domains, subunits or confirmations ofHER3. In such a case, the antigenic determinants, epitopes, parts,domains or subunits of HER3 to which the amino acid sequences and/orpolypeptides of the invention bind may be essentially the same (forexample, if HER3 contains repeated structural motifs or occurs in amultimeric form) or may be different (and in the latter case, the aminoacid sequences and polypeptides of the invention may bind to suchdifferent antigenic determinants, epitopes, parts, domains, subunits ofHER3 with an affinity and/or specificity which may be the same ordifferent). Also, for example, when HER3 exists in an activatedconformation and in an inactive conformation, the amino acid sequencesand polypeptides of the invention may bind to either one of theseconfirmation, or may bind to both these confirmations (i.e. with anaffinity and/or specificity which may be the same or different). Also,for example, the amino acid sequences and polypeptides of the inventionmay bind to a conformation of HER3 in which it is bound to a pertinentligand, may bind to a conformation of HER3 in which it not bound to apertinent ligand, or may bind to both such conformations (again with anaffinity and/or specificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides ofthe invention will generally bind to all naturally occurring orsynthetic analogs, variants, mutants, alleles, parts and fragments ofHER3; or at least to those analogs, variants, mutants, alleles, partsand fragments of HER3 that contain one or more antigenic determinants orepitopes that are essentially the same as the antigenic determinant(s)or epitope(s) to which the amino acid sequences and polypeptides of theinvention bind in HER3 (e.g. in wild-type HER3). Again, in such a case,the amino acid sequences and polypeptides of the invention may bind tosuch analogs, variants, mutants, alleles, parts and fragments with anaffinity and/or specificity that are the same as, or that are differentfrom (i.e. higher than or lower than), the affinity and specificity withwhich the amino acid sequences of the invention bind to (wild-type)HER3. It is also included within the scope of the invention that theamino acid sequences and polypeptides of the invention bind to someanalogs, variants, mutants, alleles, parts and fragments of HER3, butnot to others.

When HER3 exists in a monomeric form and in one or more multimericforms, it is within the scope of the invention that the amino acidsequences and polypeptides of the invention only bind to HER3 inmonomeric form, only bind to HER3 in multimeric form, or bind to boththe monomeric and the multimeric form. Again, in such a case, the aminoacid sequences and polypeptides of the invention may bind to themonomeric form with an affinity and/or specificity that are the same as,or that are different from (i.e. higher than or lower than), theaffinity and specificity with which the amino acid sequences of theinvention bind to the multimeric form.

Also, when HER3 can associate with other proteins or polypeptides toform protein complexes (e.g. with MET, HER1, HER2, or HER4), it iswithin the scope of the invention that the amino acid sequences andpolypeptides of the invention bind to HER3 in its non-associated state,bind to HER3 in its associated state, or bind to both, preferably bindsonly or preferentially to its non-associated state and prevents in anyevent heterodimerization at least partially. In all these cases, theamino acid sequences and polypeptides of the invention may bind to suchmultimers or associated protein complexes with an affinity and/orspecificity that may be the same as or different from (i.e. higher thanor lower than) the affinity and/or specificity with which the amino acidsequences and polypeptides of the invention bind to HER3 in itsmonomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptidesthat contain two or more amino acid sequences directed against HER3 maybind with higher avidity to HER3 than the corresponding monomeric aminoacid sequence(s). For example, and without limitation, proteins orpolypeptides that contain two or more amino acid sequences directedagainst different epitopes of HER3 may (and usually will) bind withhigher avidity than each of the different monomers, and proteins orpolypeptides that contain two or more amino acid sequences directedagainst HER3 may (and usually will) bind also with higher avidity to amultimer of HER3.

Generally, amino acid sequences and polypeptides of the invention willat least bind to those forms of HER3 (including monomeric, multimericand associated forms) that are the most relevant from a biologicaland/or therapeutic point of view, as will be clear to the skilled personand are in a preferred aspect as described herein.

It is also within the scope of the invention to use parts, fragments,analogs, mutants, variants, alleles and/or derivatives of the amino acidsequences and polypeptides of the invention, and/or to use proteins orpolypeptides comprising or essentially consisting of one or more of suchparts, fragments, analogs, mutants, variants, alleles and/orderivatives, as long as these are suitable for the uses envisagedherein. Such parts, fragments, analogs, mutants, variants, allelesand/or derivatives will usually contain (at least part of) a functionalantigen-binding site for binding against HER3; and more preferably willbe capable of specific binding to HER3, and even more preferably capableof binding to HER3 with an affinity (suitably measured and/or expressedas a K_(D)-value (actual or apparent), a K_(A)-value (actual orapparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as anIC₅₀ value, as further described herein) that is as defined herein. Somenon-limiting examples of such parts, fragments, analogs, mutants,variants, alleles, derivatives, proteins and/or polypeptides will becomeclear from the further description herein. Additional fragments orpolypeptides of the invention may also be provided by suitably combining(i.e. by linking or genetic fusion) one or more (smaller) parts orfragments as described herein.

In one specific, but non-limiting aspect of the invention, which will befurther described herein, such analogs, mutants, variants, alleles,derivatives have an increased half-life in serum (as further describedherein) compared to the amino acid sequence from which they have beenderived. For example, an amino acid sequence of the invention may belinked (chemically or otherwise) to one or more groups or moieties thatextend the half-life (such as PEG), so as to provide a derivative of anamino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of theinvention may be an amino acid sequence that comprises an immunoglobulinfold or may be an amino acid sequence that, under suitable conditions(such as physiological conditions) is capable of forming animmunoglobulin fold (i.e. by folding). Reference is inter alia made tothe review by Halaby et al., J. (1999) Protein Eng. 12, 563-71.Preferably, when properly folded so as to form an immunoglobulin fold,such an amino acid sequence is capable of specific binding (as definedherein) to HER3; and more preferably capable of binding to HER3 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein. Also, parts, fragments, analogs,mutants, variants, alleles and/or derivatives of such amino acidsequences are preferably such that they comprise an immunoglobulin foldor are capable for forming, under suitable conditions, an immunoglobulinfold.

In particular, but without limitation, the amino acid sequences of theinvention may be amino acid sequences that essentially consist of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively); or any suitablefragment of such an amino acid sequence (which will then usually containat least some of the amino acid residues that form at least one of theCDR's, as further described herein).

The amino acid sequences of the invention may in particular be animmunoglobulin sequence or a suitable fragment thereof, and more inparticular be an immunoglobulin single variable domain sequence or asuitable fragment thereof, such as light chain variable domain sequence(e.g. a V_(L)-sequence) or a suitable fragment thereof; or a heavy chainvariable domain sequence (e.g. a V_(H)-sequence) or a suitable fragmentthereof. When the amino acid sequence of the invention is a heavy chainvariable domain sequence, it may be a heavy chain variable domainsequence that is derived from a conventional four-chain antibody (suchas, without limitation, a V_(H) sequence that is derived from a humanantibody) or be a so-called V_(HH)-sequence (as defined herein) that isderived from a so-called “heavy chain antibody” from an animal of thefamily of camelids such as e.g. a llama (as defined herein).

However, it should be noted that the invention is not limited as to theorigin of the amino acid sequence of the invention (or of the nucleotidesequence of the invention used to express it), nor as to the way thatthe amino acid sequence or nucleotide sequence of the invention is (orhas been) generated or obtained. Thus, the amino acid sequences of theinvention may be naturally occurring amino acid sequences (from anysuitable species) or synthetic or semi-synthetic amino acid sequences.In a specific but non-limiting aspect of the invention, the amino acidsequence is a naturally occurring immunoglobulin sequence (from anysuitable species) or a synthetic or semi-synthetic immunoglobulinsequence, including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences or Nanobodies), “camelized” (as definedherein) immunoglobulin sequences, as well as immunoglobulin sequencesthat have been sequence optimized for optimal expression and/orstability and/or solubility, as well as immunoglobulin sequences thathave been obtained by techniques such as affinity maturation (forexample, starting from synthetic, random or naturally occurringimmunoglobulin sequences), CDR grafting, veneering, combining fragmentsderived from different immunoglobulin sequences, PCR assembly usingoverlapping primers, and similar techniques for engineeringimmunoglobulin sequences well known to the skilled person; or anysuitable combination of any of the foregoing. Reference is for examplemade to the standard handbooks, as well as to the further descriptionand prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturallyoccurring nucleotide sequences or synthetic or semi-synthetic sequences,and may for example be sequences that are isolated by PCR from asuitable naturally occurring template (e.g. DNA or RNA isolated from acell), nucleotide sequences that have been isolated from a library (andin particular, an expression library), nucleotide sequences that havebeen prepared by introducing mutations into a naturally occurringnucleotide sequence (using any suitable technique known per se, such asmismatch PCR), nucleotide sequence that have been prepared by PCR usingoverlapping primers, or nucleotide sequences that have been preparedusing techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be aimmunoglobulin singe variable domain (or an immunoglobulin singevariable domain that is suitable for use as an immunoglobulin singevariable domain), a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody), a single domain antibody (or anamino acid sequence that is suitable for use as a single domainantibody), a “dAb” (or an amino acid sequence that is suitable for useas a dAb) or a Nanobody (as defined herein, and including but notlimited to a V_(HH) sequence); other single variable domains, or anysuitable fragment of any one thereof. For a general description of(single) domain antibodies, reference is also made to the prior artcited above, as well as to EP 0 368 684. For the term “dAb's”, referenceis for example made to Ward et al. (Nature, 1989 Oct. 12; 341 (6242):544-6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; aswell as to for example WO 06/030220, WO 06/003388 and other publishedpatent applications of Domantis Ltd. It should also be noted that,although less preferred in the context of the present invention becausethey are not of mammalian origin, single domain antibodies or singlevariable domains can be derived from certain species of shark (forexample, the so-called “IgNAR domains”, see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be animmunoglobulin singe variable domain or Nanobody (as defined herein) ora suitable fragment thereof. Such Nanobodies directed against HER3 willalso be referred to herein as “Nanobodies of the invention”.

For a general description of immunoglobulin singe variable domain orNanobodies (Note: the term “immunoglobulin singe variable domain” and“Nanobodies” are used interchangeably in this application), reference ismade to the further description below, as well as to the prior art citedherein. In particular, the term Nanobody is as defined in WO 08/020,079or WO 09/068,627, and as described therein generally refers to animmunoglobulin heavy chain variable domain that has the functionaland/or structural characteristics of a V_(HH) domain (e.g. a V_(H)domain from the “heavy-chain only” antibodies that occur in Camelids),and as such may in particular be a (native) V_(HH), a humanized V_(HH)or a camelized V_(H), such as a camelized human V_(H).

In this respect, it should however be noted that this description andthe prior art mainly described Nanobodies of the so-called “V_(H)3class” (i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29),which Nanobodies form a preferred aspect of this invention. It shouldhowever be noted that the invention in its broadest sense generallycovers any type of Nanobody directed against HER3, and for example alsocovers the Nanobodies belonging to the so-called “V_(H)4 class” (i.e.Nanobodies with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin WO 07/118,670.

Generally, Nanobodies (in particular V_(HH) sequences and partiallyhumanized Nanobodies) can in particular be characterized by the presenceof one or more “Hallmark residues” (as described herein) in one or moreof the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequencewith the (general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which one or more of the Hallmark residuesare as further defined herein.

In particular, a Nanobody can be an amino acid sequence with the(general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which the framework sequences are as furtherdefined herein.

More in particular, a Nanobody can be an amino acid sequence with the(general) structureFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 below. In these Nanobodies, the CDR sequences are generally as    further defined herein.

TABLE B-2 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues 11 L, V; L, S, V, M, W, F, T, Q, E, A, R, G, K, Y,predominantly L N, P, I; preferably L 37 V, I, F; F⁽¹⁾, Y, V, L, A, H,S, I, W, C, N, G, D, T, usually V P, preferably F⁽¹⁾ or Y   44⁽⁸⁾ GE⁽³⁾, Q⁽³⁾, G⁽²⁾, D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferablyG⁽²⁾, E⁽³⁾ or Q⁽³⁾; most preferably G⁽²⁾ or Q⁽³⁾.   45⁽⁸⁾ L L⁽²⁾, R⁽³⁾,P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾   47⁽⁸⁾W, Y F⁽¹⁾, L⁽¹⁾ or W⁽²⁾ G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N,D; preferably W⁽²⁾, L⁽¹⁾ or F⁽¹⁾ 83 R or K; R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S,I, V, G, M, L, A, usually R D, Y, H; preferably K or R; most preferablyK 84 A, T, D; P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, predominantlyA G, E; preferably P 103  W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V,Q, P⁽⁶⁾, E, C; preferably W 104  G G, A, S, T, D, P, N, E, C, L;preferably G 108  L, M or T; Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;predominantly L preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but notexclusively, in combination with KERE or KQRE at positions 43-46.⁽²⁾Usually as GLEW at positions 44-47. ⁽³⁾Usually as KERE or KQRE atpositions 43-46, e.g. as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW orKQREG at positions 43-47. Alternatively, also sequences such as TERE(for example TEREL), TQRE (for example TQREL), KECE (for example KECELor KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (forexample RQREL, RQREF or RQREW), QERE (for example QEREG), QQRE, (forexample QQREW, QQREL or QQREF), KGRE (for example KGREG), KDRE (forexample KDREV) are possible. Some other possible, but less preferredsequences include for example DECKL and NVCEL. ⁽⁴⁾With both GLEW atpositions 44-47 and KERE or KQRE at positions 43-46. ⁽⁵⁾Often as KP orEP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾Inparticular, but not exclusively, in combination with GLEW at positions44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW, position108 is always Q in (non-humanized) V_(HH) sequences that also contain aW at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences atpositions 44-47, such as for example GVEW, EPEW, GLER, DQEW, DLEW, GIEW,ELEW, GPEW, EWLP, GPER, GLER and ELEW.

As further described herein, when the ISV's are nanobodies, they maycontain framework sequences that are generally as described on pages 258to 297 of WO 09/068,627 (incorporated herein by reference). Somespecific preferred, but non-limiting framework sequences (and preferredcombinations of the same) will be clear to the skilled person based onthe disclosure herein and for example includes the FR1, FR2, FR3 and FR4sequences (and combinations thereof) that are present in the Nanobodieslisted in Table A-1 (see also Table B-1), or variants thereof with onlya limited number (such as less than 5, for example 4, 3, 2 or only 1 pereach FR1, FR2, FR3 or FR4) of amino acid differences (as defined in WO09/068,627 and in WO 08/020,079), which may for example be humanizingsubstitutions and/or other amino acid differences that have beenintroduced for the purpose of sequence optimisation (some non-limitingexamples of both the former and the latter will be clear to the skilledperson based on the disclosure herein and in WO 09/068,627 and in WO08/020,079).

Thus, the invention also relates to such Nanobodies that can bind to (asdefined herein) and/or are directed against HER3, to suitable fragmentsthereof, as well as to polypeptides that comprise or essentially consistof one or more of such Nanobodies and/or suitable fragments.

SEQ ID NO's: 12 to 26 (see Table A-1) give the amino acid sequences of anumber of immunoglobulin single variable domains that have been raisedagainst HER3.

TABLE A-1 Preferred Immunoglobulin single variable domains or Nanobodysequences (also referred herein as a sequence with a particular name orSEQ ID NO: X, wherein X is a number referring to the relevant amino acidsequence): SEQ ID NO: X, wherein Name X = Amino acid sequence 18F05 12EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTQVTVSS 17B05 13EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSS 18B05 14EVQLVESGGGLVQAGGSLRLSCAASGLTFGSAPMGWYRQAPGKERELVAYISGDERIWYGDSVKGRFTISRDTTKNTLYLQMNSLKPE DTAVYYCVSDVKVRHWGQGTQVTVSS04C07 15 EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLK PEDTAVYYCLRDLNNRGQGTQVTVSS18G11 16 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSS 18E08 17EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKQRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSS 34C07 18EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSS 05A09 19EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCISSSDGSTVYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAAERRRGYSDLCRFYYGMDYWGKGTQVTVSS 17C08 20EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKERECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSS 21B02 21EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYTIGWFRQAPGKEREGVSCISSRDGDSYYADSVKGRFTISRDNAKNTAYLQMNSLKPEDTAVYYCAASASDYGLGLELFHDEYNYWGQGTQVTVSS 21F06 22EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSS 23F05 23EVQLVESGGGLVQAGGSLRLSCAASGFTFDGYAIGWFRQAPGKEREGVSCISGGDGRSYYADSVKGRFTVSSDNAKNTLYLEMNSLKPEDTAVYYCAVIWGPYCSDSYEYLYEYDYWGQGTQVTVSS 34A04 24EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYTIGWFRQAPGKEREEISCISNNDGSTYYTNSVKGRFTISSDNAKNTVYLQMNSLKPEDTAVYYCAASPHGCWYDLIPLQADFGSWGQGTQVTVSS 17E08 25EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSS 04F10 26EVQLVESGGGLVQPGGSLKLSCVASGSMFRFYHMAWYRQAPGEQRELVARIYTGGDTIYGDSVLGRFTISRDNSKNTVYLQMNTLKPE DTGVYYCNAFREYHIWGQGTQVTVSS

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined        herein) HER3 and that have at least 80%, preferably at least        85%, such as 90% or 95% or more sequence identity with at least        one of the amino acid sequences of SEQ ID NO's: 12 to 26 (see        Table A-1). These amino acid sequences may further be such that        they neutralize binding of the cognate ligand to HER3; and/or        compete with the cognate ligand for binding to HER3; and/or are        directed against the heterodimerization site (as defined herein)        on HER3;    -   amino acid sequences that cross-block (as defined herein) the        binding of at least one of the amino acid sequences of SEQ ID        NO's: 12 to 26 (see Table A-1) to HER3 and/or that compete with        at least one of the amino acid sequences of SEQ ID NO's: 12 to        26 (see Table A-1) for binding to HER3. Again, these amino acid        sequences may further be such that they neutralize binding of        the cognate ligand to HER3; and/or compete with the cognate        ligand for binding to HER3; and/or are directed against the        heterodimerization site (as defined herein) on HER3;

which amino acid sequences may be as further described herein (and mayfor example be Nanobodies); as well as polypeptides of the inventionthat comprise one or more of such amino acid sequences (which may be asfurther described herein, and may for example be bispecific and/orbiparatopic polypeptides as described herein), and nucleic acidsequences that encode such amino acid sequences and polypeptides. Suchamino acid sequences and polypeptides do not include any naturallyoccurring ligands.

In some other specific aspects, the invention provides:

-   -   amino acid sequences of the invention that are specific for HER3        compared to HER1, HER2 and/or HER4;        which amino acid sequences of the invention may be as further        described herein (and may for example be Nanobodies); as well as        polypeptides of the invention that comprise one or more of such        amino acid sequences (which may be as further described herein,        and may for example be bispecific and/or biparatopic        polypeptides as described herein), and nucleic acid sequences        that encode such amino acid sequences and polypeptides. Such        amino acid sequences and polypeptides do not include any        naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention areNanobodies which can bind (as further defined herein) to and/or aredirected against to HER3 and which:

-   i) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 12 to 26 (see Table A-1), in    which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded. In this respect, reference is also made to Table B-1,    which lists the framework 1 sequences (SEQ ID NO's: 42 to 56),    framework 2 sequences (SEQ ID NO's: 72 to 86), framework 3 sequences    (SEQ ID NO's: 102 to 116) and framework 4 sequences (SEQ ID NO's:    132 to 146) of the Nanobodies of SEQ ID NO's: 12 to 26 (see Table    A-1) (with respect to the amino acid residues at positions 1 to 4    and 27 to 30 of the framework 1 sequences, reference is also made to    the comments made below. Thus, for determining the degree of amino    acid identity, these residues are preferably disregarded);    and in which:-   ii) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 supra.

In these Nanobodies, the CDR sequences are generally as further definedherein.

Again, such Nanobodies may be derived in any suitable manner and fromany suitable source, and may for example be naturally occurring V_(HH)sequences (i.e. from a suitable species of Camelid) or synthetic orsemi-synthetic amino acid sequences, including but not limited to“humanized” (as defined herein) Nanobodies, “camelized” (as definedherein) immunoglobulin sequences (and in particular camelized heavychain variable domain sequences), as well as Nanobodies that have beenobtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing as further described herein. Also, when a Nanobodycomprises a V_(HH) sequence, said Nanobody may be suitably humanized, asfurther described herein, so as to provide one or more further(partially or fully) humanized Nanobodies of the invention. Similarly,when a Nanobody comprises a synthetic or semi-synthetic sequence (suchas a partially humanized sequence), said Nanobody may optionally befurther suitably humanized, again as described herein, again so as toprovide one or more further (partially or fully) humanized Nanobodies ofthe invention.

In particular, humanized Nanobodies may be amino acid sequences that areas generally defined for Nanobodies in the previous paragraphs, but inwhich at least one amino acid residue is present (and in particular, inat least one of the framework residues) that is and/or that correspondsto a humanizing substitution (as defined herein). Some preferred, butnon-limiting humanizing substitutions (and suitable combinationsthereof) will become clear to the skilled person based on the disclosureherein. In addition, or alternatively, other potentially usefulhumanizing substitutions can be ascertained by comparing the sequence ofthe framework regions of a naturally occurring V_(HH) sequence with thecorresponding framework sequence of one or more closely related humanV_(H) sequences, after which one or more of the potentially usefulhumanizing substitutions (or combinations thereof) thus determined canbe introduced into said V_(HH) sequence (in any manner known per se, asfurther described herein) and the resulting humanized V_(HH) sequencescan be tested for affinity for the target, for stability, for ease andlevel of expression, and/or for other desired properties. In this way,by means of a limited degree of trial and error, other suitablehumanizing substitutions (or suitable combinations thereof) can bedetermined by the skilled person based on the disclosure herein. Also,based on the foregoing, (the framework regions of) a Nanobody may bepartially humanized or fully humanized.

Some particularly preferred sequence optimized Nanobodies of theinvention are sequence optimized variants of the Nanobodies of SEQ IDNO's: 12 to 26 (see Table A-1) are some especially preferred examples.

Thus, some other preferred Nanobodies of the invention are Nanobodieswhich can bind (as further defined herein) to HER3 and which:

-   i) are a humanized variant of one of the amino acid sequences of SEQ    ID NO's: 12 to 26 (see Table A-1); and/or-   ii) have at least 80% amino acid identity with at least one of the    amino acid sequences of SEQ ID NO's: 12 to 26 (see Table A-1), in    which for the purposes of determining the degree of amino acid    identity, the amino acid residues that form the CDR sequences are    disregarded;    and in which:-   i) preferably one or more of the amino acid residues at positions    11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2 supra.

According to another specific aspect of the invention, the inventionprovides a number of stretches of amino acid residues (i.e. smallpeptides) that are particularly suited for binding to HER3. Thesestretches of amino acid residues may be present in, and/or may becorporated into, an amino acid sequence of the invention, in particularin such a way that they form (part of) the antigen binding site of anamino acid sequence of the invention. As these stretches of amino acidresidues were first generated as CDR sequences of heavy chain antibodiesor V_(HH) sequences that were raised against HER3 (or may be based onand/or derived from such CDR sequences, as further described herein),they will also generally be referred to herein as “CDR sequences” (i.e.as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). Itshould however be noted that the invention in its broadest sense is notlimited to a specific structural role or function that these stretchesof amino acid residues may have in an amino acid sequence of theinvention, as long as these stretches of amino acid residues allow theamino acid sequence of the invention to bind to HER3. Thus, generally,the invention in its broadest sense comprises any amino acid sequencethat is capable of binding to HER3 and that comprises one or more CDRsequences as described herein, and in particular a suitable combinationof two or more such CDR sequences, that are suitably linked to eachother via one or more further amino acid sequences, such that the entireamino acid sequence forms a binding domain and/or binding unit that iscapable of binding to HER3. It should however also be noted that thepresence of only one such CDR sequence in an amino acid sequence of theinvention may by itself already be sufficient to provide an amino acidsequence of the invention that is capable of binding to HER3; referenceis for example again made to the so-called “Expedite fragments”described in WO 03/050531 or WO2009/127691.

Thus, in another specific, but non-limiting aspect, the amino acidsequence of the invention may be an amino acid sequence that comprisesat least one amino acid sequence that is chosen from the groupconsisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences thatare described herein (or any suitable combination thereof). Inparticular, an amino acid sequence of the invention may be an amino acidsequence that comprises at least one antigen binding site, wherein saidantigen binding site comprises at least one amino acid sequence that ischosen from the group consisting of the CDR1 sequences, CDR2 sequencesand CDR3 sequences that are described herein (or any suitablecombination thereof).

Generally, in this aspect of the invention, the amino acid sequence ofthe invention may be any amino acid sequence that comprises at least onestretch of amino acid residues, in which said stretch of amino acidresidues has an amino acid sequence that corresponds to the sequence ofat least one of the CDR sequences described herein. Such an amino acidsequence may or may not comprise an immunoglobulin fold. For example,and without limitation, such an amino acid sequence may be a suitablefragment of an immunoglobulin sequence that comprises at least one suchCDR sequence, but that is not large enough to form a (complete)immunoglobulin fold (reference is for example again made to the“Expedite fragments” described in WO 03/050531 or WO2009/127691).Alternatively, such an amino acid sequence may be a suitable “proteinscaffold” that comprises least one stretch of amino acid residues thatcorresponds to such a CDR sequence (i.e. as part of its antigen bindingsite). Suitable scaffolds for presenting amino acid sequences will beclear to the skilled person, and for example comprise, withoutlimitation, to binding scaffolds based on or derived fromimmunoglobulins (i.e. other than the immunoglobulin sequences alreadydescribed herein), protein scaffolds derived from protein A domains(such as Affibodies™) tendamistat, fibronectin, lipocalin, CTLA-4,T-cell receptors, designed ankyrin repeats, avimers and PDZ domains(Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moietiesbased on DNA or RNA including but not limited to DNA or RNA aptamers(Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

Again, any amino acid sequence of the invention that comprises one ormore of these CDR sequences is preferably such that it can specificallybind (as defined herein) to HER3, and more in particular such that itcan bind to HER3 with an affinity (suitably measured and/or expressed asa K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent),a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value,as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect ofthe invention may be any amino acid sequence that comprises at least oneantigen binding site, wherein said antigen binding site comprises atleast two amino acid sequences that are chosen from the group consistingof the CDR1 sequences described herein, the CDR2 sequences describedherein and the CDR3 sequences described herein, such that (i) when thefirst amino acid sequence is chosen from the CDR1 sequences describedherein, the second amino acid sequence is chosen from the CDR2 sequencesdescribed herein or the CDR3 sequences described herein; (ii) when thefirst amino acid sequence is chosen from the CDR2 sequences describedherein, the second amino acid sequence is chosen from the CDR1 sequencesdescribed herein or the CDR3 sequences described herein; or (iii) whenthe first amino acid sequence is chosen from the CDR3 sequencesdescribed herein, the second amino acid sequence is chosen from the CDR1sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention maybe amino acid sequences that comprise at least one antigen binding site,wherein said antigen binding site comprises at least three amino acidsequences that are chosen from the group consisting of the CDR1sequences described herein, the CDR2 sequences described herein and theCDR3 sequences described herein, such that the first amino acid sequenceis chosen from the CDR1 sequences described herein, the second aminoacid sequence is chosen from the CDR2 sequences described herein, andthe third amino acid sequence is chosen from the CDR3 sequencesdescribed herein. Preferred combinations of CDR1, CDR2 and CDR3sequences will become clear from the further description herein. As willbe clear to the skilled person, such an amino acid sequence ispreferably an immunoglobulin sequence (as further described herein), butit may for example also be any other amino acid sequence that comprisesa suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates toan amino acid sequence directed against HER3, that comprises one or morestretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;    or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more aminoacid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence    according to b) and/or c) is preferably, and compared to the    corresponding amino acid sequence according to a), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to b) and/or c) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to a);    and/or-   iii) the amino acid sequence according to b) and/or c) may be an    amino acid sequence that is derived from an amino acid sequence    according to a) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one ormore amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence    according to e) and/or f) is preferably, and compared to the    corresponding amino acid sequence according to d), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to e) and/or f) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to d);    and/or-   iii) the amino acid sequence according to e) and/or f) may be an    amino acid sequence that is derived from an amino acid sequence    according to d) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention containsone or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence    according to h) and/or i) is preferably, and compared to the    corresponding amino acid sequence according to g), a conservative    amino acid substitution, (as defined herein);    and/or-   ii) the amino acid sequence according to h) and/or i) preferably    only contains amino acid substitutions, and no amino acid deletions    or insertions, compared to the corresponding amino acid sequence    according to g);    and/or-   iii) the amino acid sequence according to h) and/or i) may be an    amino acid sequence that is derived from an amino acid sequence    according to g) by means of affinity maturation using one or more    techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs alsogenerally apply to any amino acid sequences of the invention thatcomprise one or more amino acid sequences according to b), c), e), f),h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprisesone or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 57 to 71;-   ii) the amino acid sequences of SEQ ID NO's: 87 to 101; and-   iii) the amino acid sequences of SEQ ID NO's: 117 to 131;    or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of saidstretches of amino acid residues forms part of the antigen binding sitefor binding against HER3.

In a more specific, but again non-limiting aspect, the invention relatesto an amino acid sequence directed against HER3, that comprises two ormore stretches of amino acid residues chosen from the group consistingof:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;-   amino acid sequences that have 3, 2, or 1 amino acid difference with    at least one of the amino acid sequences of SEQ ID NO's: 87 to 101;-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;    such that (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences according to a), b)    or c), the second stretch of amino acid residues corresponds to one    of the amino acid sequences according to d), e), f), g), h) or    i); (ii) when the first stretch of amino acid residues corresponds    to one of the amino acid sequences according to d), e) or f), the    second stretch of amino acid residues corresponds to one of the    amino acid sequences according to a), b), c), g), h) or i); or (iii)    when the first stretch of amino acid residues corresponds to one of    the amino acid sequences according to g), h) or i), the second    stretch of amino acid residues corresponds to one of the amino acid    sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprisestwo or more stretches of amino acid residues chosen from the groupconsisting of:

-   i) the amino acid sequences of SEQ ID NO's: 57 to 71;-   ii) the amino acid sequences of SEQ ID NO's: 87 to 101; and-   iii) the amino acid sequences of SEQ ID NO's: 117 to 131;    such that, (i) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 57 to    71, the second stretch of amino acid residues corresponds to one of    the amino acid sequences of SEQ ID NO's: 87 to 101 or of SEQ ID    NO's: 117 to 131; (ii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 87 to    101, the second stretch of amino acid residues corresponds to one of    the amino acid sequences of SEQ ID NO's: 57 to 71 or of SEQ ID NO's:    117 to 131; or (iii) when the first stretch of amino acid residues    corresponds to one of the amino acid sequences of SEQ ID NO's: 117    to 131, the second stretch of amino acid residues corresponds to one    of the amino acid sequences of SEQ ID NO's: 57 to 71 or of SEQ ID    NO's: 87 to 101.

Also, in such an amino acid sequence, the at least two stretches ofamino acid residues again preferably form part of the antigen bindingsite for binding against HER3.

In an even more specific, but non-limiting aspect, the invention relatesto an amino acid sequence directed against HER3, that comprises three ormore stretches of amino acid residues, in which the first stretch ofamino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;    the second stretch of amino acid residues is chosen from the group    consisting of:-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;    and the third stretch of amino acid residues is chosen from the    group consisting of:-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131.

Preferably, in this specific aspect, the first stretch of amino acidresidues is chosen from the group consisting of the amino acid sequencesof SEQ ID NO's: 57 to 71; the second stretch of amino acid residues ischosen from the group consisting of the amino acid sequences of SEQ IDNO's: 87 to 101; and the third stretch of amino acid residues is chosenfrom the group consisting of the amino acid sequences of SEQ ID NO's:117 to 131.

Again, preferably, in such an amino acid sequence, the at least threestretches of amino acid residues forms part of the antigen binding sitefor binding against HER3.

Preferred combinations of such stretches of amino acid sequences willbecome clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least70% amino acid identity, preferably at least 80% amino acid identity,more preferably at least 90% amino acid identity, such as 95% amino acididentity or more or even essentially 100% amino acid identity with theCDR sequences of at least one of the amino acid sequences of SEQ IDNO's: 12 to 26 (see Table A-1). This degree of amino acid identity canfor example be determined by determining the degree of amino acididentity (in a manner described herein) between said amino acid sequenceand one or more of the sequences of SEQ ID NO's: 12 to 26 (see TableA-1), in which the amino acid residues that form the framework regionsare disregarded. Also, such amino acid sequences of the invention can beas further described herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to HER3; and more in particularbind to HER3 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), the amino acidsequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131.

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 57 to 71; and/or CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 87 to 101; and/orCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 117 to 131.

In particular, when the amino acid sequence of the invention essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), theamino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be suchthat CDR1 is chosen from the group consisting of the amino acidsequences of SEQ ID NO's: 57 to 71; and CDR2 is chosen from the groupconsisting of the amino acid sequences of SEQ ID NO's: 87 to 101; andCDR3 is chosen from the group consisting of the amino acid sequences ofSEQ ID NO's: 117 to 131.

Again, preferred combinations of CDR sequences will become clear fromthe further description herein.

Also, such amino acid sequences are preferably such that they canspecifically bind (as defined herein) to HER3; and more in particularbind to HER3 with an affinity (suitably measured and/or expressed as aK_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), ak_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, asfurther described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to anamino acid sequence that essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which the CDR sequences of said aminoacid sequence have at least 70% amino acid identity, preferably at least80% amino acid identity, more preferably at least 90% amino acididentity, such as 95% amino acid identity or more or even essentially100% amino acid identity with the CDR sequences of at least one of theamino acid sequences of SEQ ID NO's: 12 to 26 (see Table A-1). Thisdegree of amino acid identity can for example be determined bydetermining the degree of amino acid identity (in a manner describedherein) between said amino acid sequence and one or more of thesequences of SEQ ID NO's: 12 to 26 (see Table A-1), in which the aminoacid residues that form the framework regions are disregarded. Suchamino acid sequences of the invention can be as further describedherein.

In such an amino acid sequence of the invention, the framework sequencesmay be any suitable framework sequences, and examples of suitableframework sequences will be clear to the skilled person, for example onthe basis the standard handbooks and the further disclosure and priorart mentioned herein.

The framework sequences are preferably (a suitable combination of)immunoglobulin framework sequences or framework sequences that have beenderived from immunoglobulin framework sequences (for example, byhumanization or camelization). For example, the framework sequences maybe framework sequences derived from a light chain variable domain (e.g.a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence). In one particularly preferred aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequenceof the invention is a domain antibody (or an amino acid sequence that issuitable for use as a domain antibody); is a single domain antibody (oran amino acid sequence that is suitable for use as a single domainantibody); is a “dAb” (or an amino acid sequence that is suitable foruse as a dAb); or is a Nanobody (including but not limited to V_(HH)sequence). Again, suitable framework sequences will be clear to theskilled person, for example on the basis the standard handbooks and thefurther disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acidsequences of the invention may contain one or more of Hallmark residues(as defined herein), such that the amino acid sequence of the inventionis a Nanobody. Some preferred, but non-limiting examples of (suitablecombinations of) such framework sequences will become clear from thefurther disclosure herein.

In particular, when the ISV's of the invention are nNnobodies (asdescribed herein) the framework sequences present therein may be asgenerally described on pages 258 to 297 of WO 09/068,627 (incorporatedherein by reference). For example, they may contain one or more of thecombinations of Hallmark residues set out in Table A-5 of WO 09/068,627;and FR1, FR2, FR3 and FR4 may contain the amino acid residues set out inTable A-6, Table A-7, Table A-8 and Table A-9 of WO 09/068,627,respectively. Also, when the ISV's of the invention are Nanobodies, theymay belong to the KERE-group (see pages 281 to 284 of WO 09/068,627,with some representative FR1, FR2, FR3 and FR4 sequences for this groupgiven in Tables A-11/A-15, A-12, A-13 and A-14 of WO 09/068,627); to theGLEW-group (see pages 285 to 287 of WO 09/068,627, with somerepresentative FR1, FR2, FR3 and FR4 sequences for this group given inTables A-16/A-20, A-17, A-18 and A-19 of WO 09/068,627); or to the P, R,S 103 group (see pages 287 to 291 of WO 09/068,627, with somerepresentative FR1, FR2, FR3 and FR4 sequences for this group given inTables A-21/A-25, A-22, A-23 and A-24 of WO 09/068,627), which are allas described in WO 09/068,627, with some representative sequences foreach of these groups given in Table A-10 of 09/068627. As also describedin WO 09/068,627, these framework sequences may contain one or moresuitable humanizing substitutions or (other) substitutions foroptimizing the sequence (see also the further disclosure herein).

Again, some particularly preferred but non-limiting FR1, FR2, FR3 andFR4 sequences (and combinations thereof) are those described in TableB-1, or suitable variants of such FR1, FR2, FR3 and FR4 sequences,respectively (for example, with less than 6, such as 1, 2, 3, 4 or 5suitable amino acid differences in such an FR1, FR2, FR3 or FR4 comparedto a framework sequence mentioned in Table B-1, in which the amino aciddifferences may be as described in WO 09/068,627) that still essentiallyretain the desired properties of Nanobodies.

Again, as generally described herein for the amino acid sequences of theinvention, it is also possible to use suitable fragments (orcombinations of fragments) of any of the foregoing, such as fragmentsthat contain one or more CDR sequences, suitably flanked by and/orlinked via one or more framework sequences (for example, in the sameorder as these CDR's and framework sequences may occur in the full-sizedimmunoglobulin sequence from which the fragment has been derived). Suchfragments may also again be such that they comprise or can form animmunoglobulin fold, or alternatively be such that they do not compriseor cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequenceas described herein (and in particular a CDR3 sequence), that is flankedon each side by (part of) a framework sequence (and in particular, partof the framework sequence(s) that, in the immunoglobulin sequence fromwhich the fragment is derived, are adjacent to said CDR sequence. Forexample, a CDR3 sequence may be preceded by (part of) a FR3 sequence andfollowed by (part of) a FR4 sequence). Such a fragment may also containa disulphide bridge, and in particular a disulphide bridge that linksthe two framework regions that precede and follow the CDR sequence,respectively (for the purpose of forming such a disulphide bridge,cysteine residues that naturally occur in said framework regions may beused, or alternatively cysteine residues may be synthetically added toor introduced into said framework regions).

In another aspect, the invention relates to a compound or construct, andin particular a protein or polypeptide (also referred to herein as a“compound of the invention” or “polypeptide of the invention”,respectively) that comprises or essentially consists of one or moreamino acid sequences of the invention (or suitable fragments thereof),and optionally further comprises one or more other groups, residues,moieties or binding units. As will become clear to the skilled personfrom the further disclosure herein, such further groups, residues,moieties, binding units or amino acid sequences may or may not providefurther functionality to the amino acid sequence of the invention(and/or to the compound or construct in which it is present) and may ormay not modify the properties of the amino acid sequence of theinvention.

For example, such further groups, residues, moieties or binding unitsmay be one or more additional amino acid sequences, such that thecompound or construct is a (fusion) protein or (fusion) polypeptide. Ina preferred but non-limiting aspect, said one or more other groups,residues, moieties or binding units are immunoglobulin sequences, and inparticular ISV's. Even more preferably, said one or more other groups,residues, moieties or binding units are chosen from the group consistingof domain antibodies, amino acid sequences that are suitable for use asa domain antibody, single domain antibodies, amino acid sequences thatare suitable for use as a single domain antibody, “dAb”'s, amino acidsequences that are suitable for use as a dAb, or Nanobodies.

For example, such one or more (such as one or two) further ISV that arepresent in a polypeptide of the invention (i.e. in addition to the oneor more ISV's against HER3) may be directed against another target thanHER3 so as to provide a “bispecific” protein or polypeptide of theinvention (i.e. a polypeptide of the invention that contains at leastone—such as one or two—immunoglobulin single variable domain that isdirected against HER3 and at least one—such as one or two—immunoglobulinsingle variable domain that is directed against another target).

For example, according to a specific but non-limiting aspect, the aminoacid sequences, constructs, proteins or polypeptides of the inventionmay have been provided with an increased half-life (as defined herein,and compared with the same construct but without the modifications madeto provide for the increased half-life, for example without thefunctionalisation/pegylation or without the serum-albumin bindingpeptide or binding domain/ISV), for example by suitablefunctionalisation (such as pegylation) and/or by including in theconstruct a moiety or binding unit that increases the half-life of theconstruct. Examples of such functionalisation, moieties or binding unitswill be clear to the skilled person and may for example includepegylation, fusion to serum albumin, or fusion to a peptide or bindingunit that can bind to a serum protein such as serum albumin.

In the latter constructs (i.e. fusion constructs comprising at leastone—such as one or two—amino acid sequence of the invention and at leastone—such as one or two—peptide or binding unit that can bind to a serumprotein such as serum albumin), the serum-albumin binding peptide orbinding domain may be any suitable serum-albumin binding peptide orbinding domain capable of increasing the half-life of the construct(compared to the same construct without the serum-albumin bindingpeptide or binding domain), and may in particular be serum albuminbinding peptides as described in WO 2008/068280 by applicant (and inparticular WO 2009/127691 and the non-prepublished U.S. application61/301,819, both by applicant), or a serum—albumin bindingimmunoglobulin single variable domain (such as a serum-albumin bindingNanobody; for example Alb-1 or a humanized version of Alb-1 such asAlb-8 (also referred to herein as Alb-11 or ALB11), for which referenceis for example made to WO 06/122787).

With respect to half-life, it should be noted that in the invention, andby using the various half-life extending techniques described herein(for example, by suitably choosing a serum-albumin binding peptideaccording to WO 2008/068280, WO 2009/127691 and/or the non-prepublishedU.S. application 61/301,819), the half-life of a construct orpolypeptide of the invention can (and preferably is) suitably “tailored”for the intended (therapeutic and/or diagnostic) application and/or toobtain the best balance between the desired therapeutic and/orpharmacological effect and possible undesired side-effects.

Thus, for example, and without limitation, a preferred aspect of theinvention provides a “bispecific” polypeptide consisting essentially ofone immunoglobulin single variable domain directed against human HER3(or, alternatively, of two immunoglobulin single variable domainsdirected against human HER3, which may be the same or different, i.e. soas to provide—when they are the same or different—a “bivalent”polypeptide of the invention, or—when they are different—“biparatopic”polypeptide of the invention) and one immunoglobulin single variabledomain directed against human serum albumin linked by a peptide linker(as defined herein), so as to provide a bispecific polypeptide of theinvention, respectively, all as described herein. Such a protein orpolypeptide may also be in essentially isolated form (as definedherein).

In another specific, but non-limiting aspect, an amino acid sequence(such as a Nanobody) of the invention or a polypeptide of the invention(such as a bivalent, biparatopic or bispecific polypeptide of theinvention) may be suitably linked (again, chemically or via one or moresuitable linkers or spacers) to a toxin or to a (cyto)toxic residue,moiety or payload. Examples of suitable (cyto)toxic moieties, compounds,payloads or residues which can be linked to amino acids sequences orpolypeptides of the invention to provide—for example—a cytotoxiccompound (i.e. an antibody-drug conjugate or “ADC” based upon an aminoacid sequence or polypeptide of the invention) will be clear to theskilled person. Reference is for example made to the review by Ducry andStump, Bioconjugate Chem., 2010, 21 (1), pp 5-13. Such cytotoxic aminoacid sequences or polypeptides of the invention may in particular beuseful/suitable for those applications in which it is intended to kill acell that expresses the target against which the amino acid sequences orpolypeptides of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell. Usually, but without limitation, (cyto)toxic polypeptides of theinvention will either not be half-life extended or will have only alimited and/or tightly controlled half-life extension.

Alternatively, such one or more further groups, residues, moieties orbinding units that may be present in a polypeptide of the invention mayfor example be chemical groups, residues, moieties, which may or may notby themselves be biologically and/or pharmacologically active. Forexample, and without limitation, such groups may be linked to the one ormore amino acid sequences of the invention so as to provide a“derivative” of an amino acid sequence or polypeptide of the invention,as further described herein.

Also within the scope of the present invention are compounds orconstructs, that comprises or essentially consists of one or morederivatives as described herein, and optionally further comprises one ormore other groups, residues, moieties or binding units, optionallylinked via one or more linkers. Preferably, said one or more othergroups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more aminoacid sequences of the invention and the one or more groups, residues,moieties or binding units may be linked directly to each other and/orvia one or more suitable linkers or spacers. For example, when the oneor more groups, residues, moieties or binding units are amino acidsequences, the linkers may also be amino acid sequences, so that theresulting compound or construct is a fusion (protein) or fusion(polypeptide).

As will be clear from the further description above and herein, thismeans that the amino acid sequences of the invention can be used as“building blocks” to form polypeptides of the invention, i.e. bysuitably combining them with other groups, residues, moieties or bindingunits, in order to form compounds or constructs as described herein(such as, without limitations, the biparatopic, bi/multivalent andbi/multispecific polypeptides of the invention described herein) whichcombine within one molecule one or more desired properties or biologicalfunctions.

Some specific examples of polypeptides of the invention are polypeptidesthat comprise or essentially consist of:

-   -   two amino acid sequences (and in particular and preferably        ISV's) directed against HER3, which may be the same or        different, suitably linked either directly or using one or more        suitable linkers or spacers (as described herein);    -   two HRG-blocking amino acid sequences (as defined herein, and        which may be different but which are preferably the same), and        in particular and preferably two HRG-blocking ISV's (as defined        herein, which again may be different but which are preferably        the same), suitably linked either directly or using one or more        suitable linkers or spacers (as described herein);    -   two dimerisation-blocking amino acid sequences (as defined        herein, and which may be different but which are preferably the        same), and in particular and preferably two        dimerisation-blocking ISV's (as defined herein, which again may        be different but which are preferably the same), suitably linked        either directly or using one or more suitable linkers or spacers        (as described herein);    -   two domain II-binding amino acid sequences (as defined herein,        and which may be different but which are preferably the same),        and in particular and preferably two domain II-binding ISV's (as        defined herein, which again may be different but which are        preferably the same), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one other amino acid sequence (and in particular and        preferably one other ISV) that is directed against HER3 (as        defined herein) and that is not a HRG-blocking amino acid        sequence (or ISV), suitably linked either directly or using one        or more suitable linkers or spacers (as described herein);    -   one dimerisation-blocking amino acid sequence (as defined        herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein) and one other        amino acid sequence (and in particular and preferably one other        ISV) that is directed against HER3 (as defined herein) and that        is not a dimerisation-blocking amino acid sequence (or ISV),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one domain II-binding amino acid sequence (as defined herein,        and in particular and preferably one domain II-binding ISV, also        as defined herein) and one other amino acid sequence (and in        particular and preferably one other ISV) that is directed        against HER3 (as defined herein) and that is not a domain        II-binding amino acid sequence (or ISV), suitably linked either        directly or using one or more suitable linkers or spacers (as        described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one dimerisation-blocking amino acid sequence (as        defined herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one domain II-binding amino acid sequence (as        defined herein, and in particular and preferably one domain        II-binding ISV, also as defined herein), suitably linked either        directly or using one or more suitable linkers or spacers (as        described herein);    -   one dimerisation-blocking amino acid sequence (as defined        herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein) and one        domain II-binding amino acid sequence (as defined herein, and in        particular and preferably one domain II-binding ISV, also as        defined herein), suitably linked either directly or using one or        more suitable linkers or spacers (as described herein);        and such polypeptides (of which some non-limiting examples will        be clear to the skilled person based on the disclosure herein)        form further aspects of the invention.

Also, as mentioned the amino acid sequences and polypeptides of theinvention (such as the polypeptides described above) may be half-lifeextended, for example by suitable functionalisation and/or by includingin the construct a moiety or binding unit that increases the half-lifeof the construct. Where the half-life of an amino acid sequence orpolypeptide is extended by fusion to a peptide or binding unit that canbind to a serum protein such as serum albumin, the resultingconstruct/polypeptide of the invention may for example and withoutlimitation comprise or essentially consist of:

-   -   one amino acid sequence (and in particular and preferably ISV)        directed against HER3 and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein), and in        particular and preferably one HRG-blocking ISV (as defined        herein), and a group, residue, moiety or binding unit that        increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one dimerisation-blocking amino acid sequence (as defined        herein), and in particular and preferably one        dimerisation-blocking ISV (as defined herein), and a group,        residue, moiety or binding unit that increases the half-life of        said amino acid sequence (and preferably an ISV that is directed        to a serum protein and in particular to serum albumin, or a        peptide that is directed to a serum protein and in particular to        serum albumin), suitably linked either directly or using one or        more suitable linkers or spacers (as described herein);    -   one domain II-binding amino acid sequence (as defined herein),        and in particular and preferably one domain II-binding ISV (as        defined herein), and a group, residue, moiety or binding unit        that increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two amino acid sequences (and in particular and preferably        ISV's) directed against HER3, which may be the same or        different, and a group, residue, moiety or binding unit that        increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two HRG-blocking amino acid sequences (as defined herein, and        which may be different but which are preferably the same), and        in particular and preferably two HRG-blocking ISV's (as defined        herein, which again may be different but which are preferably        the same), and a group, residue, moiety or binding unit that        increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two dimerisation-blocking amino acid sequences (as defined        herein, and which may be different but which are preferably the        same), and in particular and preferably two        dimerisation-blocking ISV's (as defined herein, which again may        be different but which are preferably the same), and a group,        residue, moiety or binding unit that increases the half-life of        said amino acid sequence (and preferably an ISV that is directed        to a serum protein and in particular to serum albumin, or a        peptide that is directed to a serum protein and in particular to        serum albumin), suitably linked either directly or using one or        more suitable linkers or spacers (as described herein);    -   two domain II-binding amino acid sequences (as defined herein,        and which may be different but which are preferably the same),        and in particular and preferably two domain II-binding ISV's (as        defined herein, which again may be different but which are        preferably the same), and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one other amino acid sequence (and in particular and        preferably one other ISV) that is directed against HER3 (as        defined herein) and that is not a HRG-blocking amino acid        sequence (or ISV), and a group, residue, moiety or binding unit        that increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one dimerisation-blocking amino acid sequence (as defined        herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein) and one other        amino acid sequence (and in particular and preferably one other        ISV) that is directed against HER3 (as defined herein) and that        is not a dimerisation-blocking amino acid sequence (or ISV), and        a group, residue, moiety or binding unit that increases the        half-life of said amino acid sequence (and preferably an ISV        that is directed to a serum protein and in particular to serum        albumin, or a peptide that is directed to a serum protein and in        particular to serum albumin), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one domain II-binding amino acid sequence (as defined herein,        and in particular and preferably one domain II-binding ISV, also        as defined herein) and one other amino acid sequence (and in        particular and preferably one other ISV) that is directed        against HER3 (as defined herein) and that is not a domain        II-binding amino acid sequence (or ISV), and a group, residue,        moiety or binding unit that increases the half-life of said        amino acid sequence (and preferably an ISV that is directed to a        serum protein and in particular to serum albumin, or a peptide        that is directed to a serum protein and in particular to serum        albumin), suitably linked either directly or using one or more        suitable linkers or spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one dimerisation-blocking amino acid sequence (as        defined herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein), and a group,        residue, moiety or binding unit that increases the half-life of        said amino acid sequence (and preferably an ISV that is directed        to a serum protein and in particular to serum albumin, or a        peptide that is directed to a serum protein and in particular to        serum albumin), suitably linked either directly or using one or        more suitable linkers or spacers (as described herein);    -   one HRG-blocking amino acid sequence (as defined herein, and in        particular and preferably one HRG-blocking ISV, also as defined        herein) and one domain II-binding amino acid sequence (as        defined herein, and in particular and preferably one domain        II-binding ISV, also as defined herein), and a group, residue,        moiety or binding unit that increases the half-life of said        amino acid sequence (and preferably an ISV that is directed to a        serum protein and in particular to serum albumin, or a peptide        that is directed to a serum protein and in particular to serum        albumin), suitably linked either directly or using one or more        suitable linkers or spacers (as described herein);    -   one dimerisation-blocking amino acid sequence (as defined        herein, and in particular and preferably one        dimerisation-blocking ISV, also as defined herein) and one        domain II-binding amino acid sequence (as defined herein, and in        particular and preferably one domain II-binding ISV, also as        defined herein), and a group, residue, moiety or binding unit        that increases the half-life of said amino acid sequence (and        preferably an ISV that is directed to a serum protein and in        particular to serum albumin, or a peptide that is directed to a        serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);        and such polypeptides (of which some non-limiting examples will        be clear to the skilled person based on the disclosure        herein—for example, some are listed in Table A-2 below) also        form further aspects of the invention.

Of these, particularly preferred are polypeptides of the invention thateither:

-   a) comprise or essentially consist of one HRG-blocking amino acid    sequence (as defined herein, and in particular and preferably one    HRG-blocking ISV, also as defined herein) and one    dimerisation-blocking amino acid sequence (as defined herein, and in    particular and preferably one dimerisation-blocking ISV, also as    defined herein), and that optionally further comprise a group,    residue, moiety or binding unit that increases the half-life of said    amino acid sequence (and preferably an ISV that is directed to a    serum protein and in particular to serum albumin such as    Alb-8/Alb-11, or a peptide that is directed to a serum protein and    in particular to serum albumin), suitably linked either directly or    using one or more suitable linkers or spacers (as described herein).    Such polypeptides may, for example and without limitation, comprise    one 17B05-like sequence, one 21F06-like sequence, and a serum    albumin binding ISV such as Alb-8, optionally suitably linked to    each other via one or more suitable spacers or linkers. A specific    preferred but non-limiting example of such polypeptide is    HER3MS00135 (SEQ ID NO:282); or that-   b) comprise or essentially consist of one dimerisation-blocking    amino acid sequence (as defined herein, and in particular and    preferably one dimerisation-blocking ISV, also as defined herein)    and one domain II-binding amino acid sequence (as defined herein,    and in particular and preferably one domain II-binding ISV, also as    defined herein), and a group, residue, moiety or binding unit that    increases the half-life of said amino acid sequence (and preferably    an ISV that is directed to a serum protein and in particular to    serum albumin such as Alb-8/Alb-11, or a peptide that is directed to    a serum protein and in particular to serum albumin), suitably linked    either directly or using one or more suitable linkers or spacers (as    described herein). Such polypeptides may, for example and without    limitation, comprise one 17B05-like sequence, one 18G11-like    sequence, and a serum albumin binding ISV such as Alb-8, optionally    suitably linked to each other via one or more suitable spacers or    linkers. Two specific preferred, but non-limiting examples of such a    polypeptide are HER3MS00212 (SEQ ID NO:319) and HER3MS00215 (SEQ ID    NO:322).

As also already mentioned herein, when one of the above polypeptides:(i) contains an HRG-blocking amino acid sequence, it is preferablyeither a 21F06-like sequence (as defined herein) or a 04C07-likesequence (also as defined herein); and/or (ii) contains adimerisation-blocking sequence, it is preferably a 17B05-like sequence(as defined herein); and/or (iii) contains a domain II-binding sequence,it is preferably either a 18G11-like sequence or a 34C07-like sequence.Some specific examples of such polypeptides of the invention arepolypeptides that comprise or essentially consist of:

-   -   two 21F06-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   two 04C07-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   two 17B05-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   two 18G11-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   two 34C07-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   one 21F06-like sequence (as defined herein) and one 04C07-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 21F06-like sequence (as defined herein) and one 17B05-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 21F06-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 21F06-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 04C07-like sequence (as defined herein) and one 17B05-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 04C07-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 04C07-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 17B05-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 17B05-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);    -   one 18G11-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), suitably linked either directly or        using one or more suitable linkers or spacers (as described        herein);        and such polypeptides (of which some non-limiting examples will        be clear to the skilled person based on the disclosure        herein—for example, some are listed in Table A-2 below) again        form further aspects of the invention.

Also, again, such polypeptides may be half-life extended, i.e. forexample by suitable functionalisation and/or by including in theconstruct a moiety or binding unit that increases the half-life of theconstruct. Where the half-life of an amino acid sequence or polypeptideis extended by fusion to a peptide or binding unit that can bind to aserum protein such as serum albumin, the resulting construct/polypeptideof the invention may for example and without limitation comprise oressentially consist of:

-   -   two 21F06-like sequences (as defined herein, and which may be        the same or different), suitably linked either directly or using        one or more suitable linkers or spacers (as described herein);    -   two 04C07-like sequences (as defined herein, and which may be        the same or different), and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two 17B05-like sequences (as defined herein, and which may be        the same or different), and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two 18G11-like sequences (as defined herein, and which may be        the same or different), and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   two 34C07-like sequences (as defined herein, and which may be        the same or different), and a group, residue, moiety or binding        unit that increases the half-life of said amino acid sequence        (and preferably an ISV that is directed to a serum protein and        in particular to serum albumin, or a peptide that is directed to        a serum protein and in particular to serum albumin), suitably        linked either directly or using one or more suitable linkers or        spacers (as described herein);    -   one 21F06-like sequence (as defined herein) and one 04C07-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 21F06-like sequence (as defined herein) and one 17B05-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 21F06-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 21F06-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 04C07-like sequence (as defined herein) and one 17B05-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 04C07-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 04C07-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 17B05-like sequence (as defined herein) and one 18G11-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 17B05-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);    -   one 18G11-like sequence (as defined herein) and one 34C07-like        sequence (as defined herein), and a group, residue, moiety or        binding unit that increases the half-life of said amino acid        sequence (and preferably an ISV that is directed to a serum        protein and in particular to serum albumin, or a peptide that is        directed to a serum protein and in particular to serum albumin),        suitably linked either directly or using one or more suitable        linkers or spacers (as described herein);        and again such polypeptides (of which some non-limiting examples        will be clear to the skilled person based on the disclosure        herein—for example, some are listed in Table A-2 below) again        form further aspects of the invention.

Some specifically preferred, but non-limiting polypeptides of theinvention comprise or essentially consist of:

-   a) one 21F06-like sequence (as defined herein) and one 17B05-like    sequence (as defined herein), and group, residue, moiety or binding    unit that increases the half-life of said amino acid sequence (and    preferably an ISV that is directed to a serum protein and in    particular to serum albumin such as Alb-8, or a peptide that is    directed to a serum protein and in particular to serum albumin),    suitably linked either directly or using one or more suitable    linkers or spacers (as described herein). A specific preferred but    non-limiting example of such polypeptide is HER3MS00135 (SEQ ID    NO:282); or-   b) one 17B05-like sequence (as defined herein) and one 18G11-like    sequence (as defined herein), and a group, residue, moiety or    binding unit that increases the half-life of said amino acid    sequence (and preferably an ISV that is directed to a serum protein    and in particular to serum albumin such as Alb-8, or a peptide that    is directed to a serum protein and in particular to serum albumin),    suitably linked either directly or using one or more suitable    linkers or spacers (as described herein). Two specific preferred,    but non-limiting examples of such a polypeptide are HER3MS00212 (SEQ    ID NO:319) and HER3MS00215 (SEQ ID NO:322).    Some specifically preferred, but non-limiting examples of    polypeptides of the invention are HER3MS00135 (SEQ ID NO:282),    HER3MS00212 (SEQ ID NO:319) and HER3MS00212 (SEQ ID NO:322). The    invention thus also relates to:-   a) the polypeptide HER3MS00135 (SEQ ID NO:282), as well as to    polypeptides that have at least 80%, such as at least 85%, for    example at least 90%, and up to 95% or more (such as 98%, 99% or    more) sequence identity with HER3MS00135 (SEQ ID NO:282), in which    the 21F06-like sequence and 17B05-like sequence present in such    polypeptides are preferably as described herein;-   b) the polypeptide HER3MS00212 (SEQ ID NO:319), as well as to    polypeptides that have at least 80%, such as at least 85%, for    example at least 90%, and up to 95% or more (such as 98%, 99% or    more) sequence identity with HER3MS00212 (SEQ ID NO:319), in which    the 18G11-like sequence and 17B05-like sequence present in such    polypeptides are preferably as described herein;-   c) the polypeptide HER3MS00215 (SEQ ID NO:322), as well as to    polypeptides that have at least 80%, such as at least 85%, for    example at least 90%, and up to 95% or more (such as 98%, 99% or    more) sequence identity with HER3MS00215 (SEQ ID NO:322), in which    the 18G11-like sequence and 17B05-like sequence present in such    polypeptides are preferably as described herein.

It has also been found that some of the polypeptides provided by theinvention may have an effect on HER3 internalization, and in particularmay increase internalisation of the HER3 receptor. This may for examplehave the effect of reducing the number of HER3 receptors present on thesurface of a cell, and thus reducing the ligand-sensitivity of the celland/or reducing the HER3 mediated signalling in/by said cell and/ormodulating other HER3 related biological effects of said cell. Referenceis for instance made to Example 17 below. This effect on HER3internalisation shown by the polypeptides of the invention isparticularly surprising because so far, none of the correspondingmonovalent building blocks present in said internalisation-promotingpolypeptides have been found to have a similar influence on HER3internalisation.

It will be clear to the skilled person that for some applications of thepolypeptides of the invention, it may be advantageous to use apolypeptide of the invention that can promote or increase HER3internalisation of a HER3 expressing cell that is exposed to orcontacted with such a polypeptide (for example, after administration ofsaid polypeptide to a patient or other subject). Thus, in one aspect, apolypeptide as described herein (which may contain any of the buildingblocks/ISV's described herein and which may for example be any of thepolypeptides described on the previous pages) is preferably such that itis capable of promoting or increasing HER3 internalisation of a cell,for example by at least 5%, such as at least 10%, for example at least25%, such as 50% or even 90% or more, as measured using a suitableassay, for example the assay of Examples 17 and 20.

It will also be clear to the skilled person that for other applicationsof the polypeptides of the invention, it may be advantageous to use apolypeptide of the invention that essentially does not alter, promote orincrease HER3 internalisation of a HER3 expressing cell that is exposedto or contacted with such a polypeptide (for example, afteradministration of said polypeptide to a patient or other subject). Thus,in one aspect, a polypeptide as described herein (which may contain anyof the building blocks/ISV's described herein and which may for examplebe any of the polypeptides described on the previous pages) ispreferably such that is does not alter or affect HER3 internalisation ofa cell, as measured using a suitable assay, for example the assay ofExamples 17 and 20.

The compounds or polypeptides of the invention can generally be preparedby a method which comprises at least one step of suitably linking theone or more amino acid sequences of the invention to the one or morefurther groups, residues, moieties or binding units, optionally via theone or more suitable linkers, so as to provide the compound orpolypeptide of the invention. Polypeptides of the invention can also beprepared by a method which generally comprises at least the steps ofproviding a nucleic acid that encodes a polypeptide of the invention,expressing said nucleic acid in a suitable manner, and recovering theexpressed polypeptide of the invention. Such methods can be performed ina manner known per se, which will be clear to the skilled person, forexample on the basis of the methods and techniques further describedherein.

The process of designing/selecting and/or preparing a compound orpolypeptide of the invention, starting from an amino acid sequence ofthe invention, is also referred to herein as “formatting” said aminoacid sequence of the invention; and an amino acid of the invention thatis made part of a compound or polypeptide of the invention is said to be“formatted” or to be “in the format of” said compound or polypeptide ofthe invention. Examples of ways in which an amino acid sequence of theinvention can be formatted and examples of such formats will be clear tothe skilled person based on the disclosure herein; and such formattedamino acid sequences form a further aspect of the invention.

In one specific aspect of the invention, a compound of the invention ora polypeptide of the invention may have an increased half-life, comparedto the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such compounds and polypeptideswill become clear to the skilled person based on the further disclosureherein, and for example comprise amino acid sequences or polypeptides ofthe invention that have been chemically modified to increase thehalf-life thereof (for example, by means of pegylation); amino acidsequences of the invention that comprise at least one additional bindingsite for binding to a serum protein (such as serum albumin); orpolypeptides of the invention that comprise at least one amino acidsequence of the invention that is linked to at least one moiety (and inparticular at least one amino acid sequence) that increases thehalf-life of the amino acid sequence of the invention. Examples ofpolypeptides of the invention that comprise such half-life extendingmoieties or amino acid sequences will become clear to the skilled personbased on the further disclosure herein; and for example include, withoutlimitation, polypeptides in which the one or more amino acid sequencesof the invention are suitable linked to one or more serum proteins orfragments thereof (such as (human) serum albumin or suitable fragmentsthereof) or to one or more binding units that can bind to serum proteins(such as, for example, domain antibodies, amino acid sequences that aresuitable for use as a domain antibody, single domain antibodies, aminoacid sequences that are suitable for use as a single domain antibody,“dAb”'s, amino acid sequences that are suitable for use as a dAb, orNanobodies that can bind to serum proteins such as serum albumin (suchas human serum albumin), serum immunoglobulins such as IgG, ortransferrine; reference is made to the further description andreferences mentioned herein); polypeptides in which an amino acidsequence of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more amino acid sequences of the invention are suitable linked toone or more small proteins or peptides that can bind to serum proteins(such as, without limitation, the proteins and peptides described in WO91/01743, WO 01/45746, WO 02/076489 and to the US provisionalapplication of Ablynx N.V. entitled “Peptides capable of binding toserum proteins” of Ablynx N.V. filed on Dec. 5, 2006 (see alsoPCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increasedhalf-life preferably have a half-life that is at least 1.5 times,preferably at least 2 times, such as at least 5 times, for example atleast 10 times or more than 20 times, greater than the half-life of thecorresponding amino acid sequence of the invention per se. For example,the compounds or polypeptides of the invention with increased half-lifemay have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se. In apreferred, but non-limiting aspect of the invention, above increases inhalf-life are achieved in mammals such as e.g. human, i.e. preferably inhumans.

In a preferred, but non-limiting aspect of the invention, such compoundsor polypeptides of the invention have a serum half-life that isincreased with more than 1 hours, preferably more than 2 hours, morepreferably more than 6 hours, such as more than 12 hours, or even morethan 24, 48 or 72 hours, compared to the corresponding amino acidsequence of the invention per se. In a preferred, but non-limitingaspect of the invention, above increases in half-life are achieved inmammals such as e.g. human, i.e. preferably in humans.

In another preferred, but non-limiting aspect of the invention, suchcompounds or polypeptides of the invention exhibit a serum half-life inhuman of at least about 12 hours, preferably at least 24 hours, morepreferably at least 48 hours, even more preferably at least 72 hours ormore. For example, compounds or polypeptides of the invention may have ahalf-life of at least 5 days (such as about 5 to 10 days), preferably atleast 9 days (such as about 9 to 14 days), more preferably at leastabout 10 days (such as about 10 to 15 days), or at least about 11 days(such as about 11 to 16 days), more preferably at least about 12 days(such as about 12 to 18 days or more), or more than 14 days (such asabout 14 to 19 days). In a preferred, but non-limiting aspect of theinvention, above serum half-lifes are achieved in mammals such as e.g.human, i.e. preferably in humans.

In another aspect, the invention relates to a nucleic acid that encodesan amino acid sequence of the invention or a polypeptide of theinvention (or a suitable fragment thereof). Such a nucleic acid willalso be referred to herein as a “nucleic acid of the invention” and mayfor example be in the form of a genetic construct, as further describedherein.

In another aspect, the invention relates to a host or host cell thatexpresses (or that under suitable circumstances is capable ofexpressing) an amino acid sequence of the invention and/or a polypeptideof the invention; and/or that contains a nucleic acid of the invention.Some preferred but non-limiting examples of such hosts or host cellswill become clear from the further description herein.

The invention further relates to a product or composition containing orcomprising at least one amino acid sequence of the invention, at leastone polypeptide of the invention (or a suitable fragment thereof) and/orat least one nucleic acid of the invention, and optionally one or morefurther components of such compositions known per se, e.g. depending onthe intended use of the composition. Such a product or composition mayfor example be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention also relates to the use of an amino acid sequence,Nanobody or polypeptide of the invention, or of a composition comprisingthe same, in (methods or compositions for) modulating HER3, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or in a multicellular organism, and in particular in amammal, and more in particular in a human being, such as in a humanbeing that is at risk of or suffers from a variety of cancers).

The invention also relates to methods for modulating HER3, either invitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an asingle cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a variety of cancers), which methodcomprises at least the step of contacting HER3 with at least one aminoacid sequence, ISV, Nanobody or polypeptide of the invention, or with acomposition comprising the same, in a manner and in an amount suitableto modulate HER3, with at least one amino acid sequence, ISV, Nanobodyor polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence,ISV, Nanobody or polypeptide of the invention in the preparation of acomposition (such as, without limitation, a pharmaceutical compositionor preparation as further described herein) for modulating HER3, eitherin vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in ana single cell or multicellular organism, and in particular in a mammal,and more in particular in a human being, such as in a human being thatis at risk of or suffers from a variety of cancers).

In the context of the present invention, “modulating” or “to modulate”generally means either reducing or inhibiting the activity of, oralternatively increasing the activity of, HER3, as measured using asuitable in vitro, cellular or in vivo assay (such as those mentionedherein). In particular, “modulating” or “to modulate” may mean eitherreducing or inhibiting the activity of, or alternatively increasing theactivity of HER3, as measured using a suitable in vitro, cellular or invivo assay (such as those mentioned herein), by at least 1%, preferablyat least 5%, such as at least 10% or at least 25%, for example by atleast 50%, at least 60%, at least 70%, at least 80%, or 90% or more,compared to activity of HER3 in the same assay under the same conditionsbut without the presence of the amino acid sequence, ISV, Nanobody orpolypeptide of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of HER3 for one ormore of its targets, heterodimerization partners, ligands or substrates;and/or effecting a change (which may either be an increase or adecrease) in the sensitivity of HER3 for one or more conditions in themedium or surroundings in which HER3 is present (such as pH, ionstrength, the presence of co-factors, etc.), compared to the sameconditions but without the presence of the amino acid sequence, ISV,Nanobody or polypeptide of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, such as the assaysdescribed herein or in the prior art cited herein.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist or as an antagonist, respectively) with respect to one or morebiological or physiological mechanisms, effects, responses, functions,pathways or activities in which HER3 (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in in vivo assay) assay known per se, such as the assays describedherein or in the prior art cited herein. In particular, an action as anagonist or antagonist may be such that an intended biological orphysiological activity is increased or decreased, respectively, by atleast 1%, preferably at least 5%, such as at least 10% or at least 25%,for example by at least 50%, at least 60%, at least 70%, at least 80%,or 90% or more, compared to the biological or physiological activity inthe same assay under the same conditions but without the presence of theamino acid sequence, ISV, Nanobody or polypeptide of the invention.

Modulating may for example involve reducing or inhibiting the binding ofHER3 to one of its substrates or ligands and/or competing with a naturalligand, substrate for binding to HER3. Modulating may also involveactivating HER3 or the mechanism or pathway in which it is involved.Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

The invention further relates to methods for preparing or generating theamino acid sequences, polypeptides, nucleic acids, host cells, productsand compositions described herein. Some preferred but non-limitingexamples of such methods will become clear from the further descriptionherein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;    and-   b) screening said set, collection or library of amino acid sequences    for amino acid sequences that can bind to and/or have affinity for    HER3;    and-   c) isolating the amino acid sequence(s) that can bind to and/or have    affinity for HER3.

In such a method, the set, collection or library of amino acid sequencesmay be any suitable set, collection or library of amino acid sequences.For example, the set, collection or library of amino acid sequences maybe a set, collection or library of immunoglobulin sequences (asdescribed herein), such as a naïve set, collection or library ofimmunoglobulin sequences; a synthetic or semi-synthetic set, collectionor library of immunoglobulin sequences; and/or a set, collection orlibrary of immunoglobulin sequences that have been subjected to affinitymaturation.

Also, in such a method, the set, collection or library of amino acidsequences may be a set, collection or library of heavy chain variabledomains (such as V_(H) domains or V_(HH) domains) or of light chainvariable domains. For example, the set, collection or library of aminoacid sequences may be a set, collection or library of domain antibodiesor single domain antibodies, or may be a set, collection or library ofamino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofamino acid sequences may be an immune set, collection or library ofimmunoglobulin sequences, for example derived from a mammal that hasbeen suitably immunized with HER3 or with a suitable antigenicdeterminant based thereon or derived therefrom, such as an antigenicpart, fragment, region, domain, loop or other epitope thereof. In oneparticular aspect, said antigenic determinant may be an extracellularpart, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acidsequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) amino acid sequences will beclear to the person skilled in the art, for example on the basis of thefurther disclosure herein. Reference is also made to the review byHoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid    sequences;-   b) screening said collection or sample of cells for cells that    express an amino acid sequence that can bind to and/or have affinity    for HER3;    and-   c) either (i) isolating said amino acid sequence; or (ii) isolating    from said cell a nucleic acid sequence that encodes said amino acid    sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulinsequence, the collection or sample of cells may for example be acollection or sample of B-cells. Also, in this method, the sample ofcells may be derived from a mammal that has been suitably immunized withHER3 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequencedirected against HER3 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for HER3;    and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

In such a method, the set, collection or library of nucleic acidsequences encoding amino acid sequences may for example be a set,collection or library of nucleic acid sequences encoding a naïve set,collection or library of immunoglobulin sequences; a set, collection orlibrary of nucleic acid sequences encoding a synthetic or semi-syntheticset, collection or library of immunoglobulin sequences; and/or a set,collection or library of nucleic acid sequences encoding a set,collection or library of immunoglobulin sequences that have beensubjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acidsequences may encode a set, collection or library of heavy chainvariable domains (such as V_(H) domains or V_(HH) domains) or of lightchain variable domains. For example, the set, collection or library ofnucleic acid sequences may encode a set, collection or library of domainantibodies or single domain antibodies, or a set, collection or libraryof amino acid sequences that are capable of functioning as a domainantibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences, for example derived from a mammal that has beensuitably immunized with HER3 or with a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for exampleencode an immune set, collection or library of heavy chain variabledomains or of light chain variable domains. In one specific aspect, theset, collection or library of nucleotide sequences may encode a set,collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to the review by Hoogenboom in NatureBiotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating an amino acid sequencedirected against HER3 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding amino acid sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode an amino acid    sequence that can bind to and/or has affinity for HER3 and that is    cross-blocked or is cross blocking a ISV or Nanobody of the    invention, e.g. SEQ ID NO: 12 to 26 (Table A-1), or a humanized ISV    or Nanobody of the invention; and-   c) isolating said nucleic acid sequence, followed by expressing said    amino acid sequence.

The invention also relates to amino acid sequences that are obtained bythe above methods, or alternatively by a method that comprises the oneof the above methods and in addition at least the steps of determiningthe nucleotide sequence or amino acid sequence of said immunoglobulinsequence; and of expressing or synthesizing said amino acid sequence ina manner known per se, such as by expression in a suitable host cell orhost organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of theinvention may be suitably humanized (or alternatively camelized); and/orthe amino acid sequence(s) thus obtained may be linked to each other orto one or more other suitable amino acid sequences (optionally via oneor more suitable linkers) so as to provide a polypeptide of theinvention. Also, a nucleic acid sequence encoding an amino acid sequenceof the invention may be suitably humanized (or alternatively camelized)and suitably expressed; and/or one or more nucleic acid sequencesencoding an amino acid sequence of the invention may be linked to eachother or to one or more nucleic acid sequences that encode othersuitable amino acid sequences (optionally via nucleotide sequences thatencode one or more suitable linkers), after which the nucleotidesequence thus obtained may be suitably expressed so as to provide apolypeptide of the invention.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with HER3. Some preferred but non-limiting applications anduses will become clear from the further description herein.

The invention also relates to the amino acid sequences, compounds,constructs, polypeptides, nucleic acids, host cells, products andcompositions described herein for use in therapy.

In particular, the invention also relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of a disease ordisorder that can be prevented or treated by administering, to a subjectin need thereof, of (a pharmaceutically effective amount of) an aminoacid sequence, compound, construct or polypeptide as described herein.

More in particular, the invention relates to the amino acid sequences,compounds, constructs, polypeptides, nucleic acids, host cells, productsand compositions described herein for use in therapy of variety ofcancers.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein, in which theinvention will be described and discussed in more detail with referenceto the Nanobodies of the invention and polypeptides of the inventioncomprising the same, which form some of the preferred aspects of theinvention.

As will become clear from the further description herein, Nanobodiesgenerally offer certain advantages (outlined herein) compared to “dAb's”or similar (single) domain antibodies or immunoglobulin sequences, whichadvantages are also provided by the Nanobodies of the invention.However, it will be clear to the skilled person that the more generalaspects of the teaching below can also be applied (either directly oranalogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their    usual meaning in the art, which will be clear to the skilled person.    Reference is for example made to the standard handbooks mentioned in    paragraph a) on page 46 of WO 08/020,079.-   b) Unless indicated otherwise, the term “immunoglobulin single    variable domain” is used as a general term to include but not    limited to antigen-binding domains or fragments such as V_(HH)    domains or V_(H) or V_(L) domains, respectively, as e.g. herein    described. The terms antigen-binding molecules or antigen-binding    protein are used interchangeably and include also the term    nanobodies. The immunoglobulin single variable domains further are    light chain variable domain sequences (e.g. a V_(L)-sequence), or    heavy chain variable domain sequences (e.g. a V_(H)-sequence); more    specifically, they can be heavy chain variable domain sequences that    are derived from a conventional four-chain antibody or heavy chain    variable domain sequences that are derived from a heavy chain    antibody. Accordingly, the immunoglobulin single variable domains    can be domain antibodies, or immunoglobulin sequences that are    suitable for use as domain antibodies, single domain antibodies, or    immunoglobulin sequences that are suitable for use as single domain    antibodies, “dAbs”, or immunoglobulin sequences that are suitable    for use as dAbs, or nanobodies, or immunoglobulin sequences that are    suitable for use as nanobodies, including but not limited to V_(HH)    sequences. The invention includes immunoglobulin sequences of    different origin, comprising mouse, rat, rabbit, donkey, shark,    human and camelid immunoglobulin sequences. The immunoglobulin    single variable domain includes fully human, humanized, otherwise    sequence optimized or chimeric immunoglobulin sequences. The    immunoglobulin single variable domain and structure of an    immunoglobulin single variable domain can be considered—without    however being limited thereto—to be comprised of four framework    regions or “FR's”, which are referred to in the art and herein as    “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as    “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,    respectively; which framework regions are interrupted by three    complementary determining regions or “CDR's”, which are referred to    in the art as “Complementarity Determining Region 1” or “CDR1”; as    “Complementarity Determining Region 2” or “CDR2”; and as    “Complementarity Determining Region 3” or “CDR3”, respectively. It    is noted that the terms nanobody or nanobodies are registered    trademarks of Ablynx N.V. and thus may also be referred to as    Nanobody® and/or Nanobodies®).-   c) Unless indicated otherwise, the terms “immunoglobulin sequence”,    “sequence”, “nucleotide sequence” and “nucleic acid” are as    described in paragraph b) on page 46 of WO 08/020,079.-   d) Unless indicated otherwise, all methods, steps, techniques and    manipulations that are not specifically described in detail can be    performed and have been performed in a manner known per se, as will    be clear to the skilled person. Reference is for example again made    to the standard handbooks and the general background art mentioned    herein and to the further references cited therein; as well as to    for example the following reviews Presta, Adv. Drug Deliv. Rev.    2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):    49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;    Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et    al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for    protein engineering, such as affinity maturation and other    techniques for improving the specificity and other desired    properties of proteins such as immunoglobulins.-   e) Amino acid residues will be indicated according to the standard    three-letter or one-letter amino acid code. Reference is made to    Table A-2 on page 48 of the International application WO 08/020,079    of Ablynx N.V. entitled “Amino acid sequences directed against IL-6R    and polypeptides comprising the same for the treatment of diseases    and disorders associated with Il-6 mediated signalling”.-   f) For the purposes of comparing two or more nucleotide sequences,    the percentage of “sequence identity” between a first nucleotide    sequence and a second nucleotide sequence may be calculated or    determined as described in paragraph e) on page 49 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of nucleotides in the first nucleotide sequence that are identical    to the nucleotides at the corresponding positions in the second    nucleotide sequence] by [the total number of nucleotides in the    first nucleotide sequence] and multiplying by [100%], in which each    deletion, insertion, substitution or addition of a nucleotide in the    second nucleotide sequence—compared to the first nucleotide    sequence—is considered as a difference at a single nucleotide    (position); or using a suitable computer algorithm or technique,    again as described in paragraph e) on pages 49 of WO 08/020,079    (incorporated herein by reference).-   g) For the purposes of comparing two or more amino acid sequences,    the percentage of “sequence identity” between a first amino acid    sequence and a second amino acid sequence (also referred to herein    as “amino acid identity”) may be calculated or determined as    described in paragraph 0 on pages 49 and 50 of WO 08/020,079    (incorporated herein by reference), such as by dividing [the number    of amino acid residues in the first amino acid sequence that are    identical to the amino acid residues at the corresponding positions    in the second amino acid sequence] by [the total number of amino    acid residues in the first amino acid sequence] and multiplying by    [100%], in which each deletion, insertion, substitution or addition    of an amino acid residue in the second amino acid sequence—compared    to the first amino acid sequence—is considered as a difference at a    single amino acid residue (position), i.e. as an “amino acid    difference” as defined herein; or using a suitable computer    algorithm or technique, again as described in paragraph f) on pages    49 and 50 of WO 08/020,079 (incorporated herein by reference).

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, as described on page 50 of WO08/020,079.

Any amino acid substitutions applied to the polypeptides describedherein may also be based on the analysis of the frequencies of aminoacid variations between homologous proteins of different speciesdeveloped by Schulz et al., Principles of Protein Structure,Springer-Verlag, 1978, on the analyses of structure forming potentialsdeveloped by Chou and Fasman, Biochemistry 13: 211, 1974 and Adv.Enzymol., 47: 45-149, 1978, and on the analysis of hydrophobicitypatterns in proteins developed by Eisenberg et al., Proc. Nad. Acad.Sci. USA 81: 140-144, 1984; Kyte & Doolittle; J. Molec. Biol. 157:105-132, 1981, and Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353,1986, all incorporated herein in their entirety by reference.Information on the primary, secondary and tertiary structure ofNanobodies is given in the description herein and in the generalbackground art cited above. Also, for this purpose, the crystalstructure of a V_(HH) domain from a llama is for example given byDesmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);Spinelli et al., Natural Structural Biology (1996); 3, 752-757; andDecanniere et al., Structure, Vol. 7, 4, 361 (1999). Further informationabout some of the amino acid residues that in conventional V_(H) domainsform the V_(H)/V_(L) interface and potential camelizing substitutions onthese positions can be found in the prior art cited above.

-   h) Amino acid sequences and nucleic acid sequences are said to be    “exactly the same” if they have 100% sequence identity (as defined    herein) over their entire length.-   i) When comparing two amino acid sequences, the term “amino acid    difference” refers to an insertion, deletion or substitution of a    single amino acid residue on a position of the first sequence,    compared to the second sequence; it being understood that two amino    acid sequences can contain one, two or more such amino acid    differences.-   j) When a nucleotide sequence or amino acid sequence is said to    “comprise” another nucleotide sequence or amino acid sequence,    respectively, or to “essentially consist of” another nucleotide    sequence or amino acid sequence, this has the meaning given in    paragraph i) on pages 51-52 of WO 08/020,079.-   k) The term “in essentially isolated form” has the meaning given to    it in paragraph j) on pages 52 and 53 of WO 08/020,079.-   l) The terms “domain” and “binding domain” have the meanings given    to it in paragraph k) on page 53 of WO 08/020,079.-   m) The terms “antigenic determinant” and “epitope”, which may also    be used interchangeably herein, have the meanings given to it in    paragraph 1) on page 53 of WO 08/020,079.-   n) As further described in paragraph m) on page 53 of WO 08/020,079,    an amino acid sequence (such as a Nanobody, an antibody, a    polypeptide of the invention, or generally an antigen binding    protein or polypeptide or a fragment thereof) that can    (specifically) bind to, that has affinity for and/or that has    specificity for a specific antigenic determinant, epitope, antigen    or protein (or for at least one part, fragment or epitope thereof)    is said to be “against” or “directed against” said antigenic    determinant, epitope, antigen or protein.-   o) The term “specificity” has the meaning given to it in    paragraph n) on pages 53-56 of WO 08/020,079; and as mentioned    therein refers to the number of different types of antigens or    antigenic determinants to which a particular antigen-binding    molecule or antigen-binding protein (such as a Nanobody or a    polypeptide of the invention) molecule can bind. The specificity of    an antigen-binding protein can be determined based on affinity    and/or avidity, as described on pages 53-56 of WO 08/020,079    (incorporated herein by reference), which also describes some    preferred techniques for measuring binding between an    antigen-binding molecule (such as a Nanobody or polypeptide of the    invention) and the pertinent antigen. Typically, antigen-binding    proteins (such as the amino acid sequences, Nanobodies and/or    polypeptides of the invention) will bind to their antigen with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10 ⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant    (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷ to    10¹² liter/moles or more and more preferably 10⁸ to 10¹²    liter/moles). Any K_(D) value greater than 10⁴ mol/liter (or any    K_(A) value lower than 10⁴ M⁻¹) liters/mol is generally considered    to indicate non-specific binding. Preferably, a monovalent    immunoglobulin sequence of the invention will bind to the desired    antigen with an affinity less than 500 nM, preferably less than 200    nM, more preferably less than 10 nM, such as less than 500 pM.    Specific binding of an antigen-binding protein to an antigen or    antigenic determinant can be determined in any suitable manner known    per se, including, for example, Scatchard analysis and/or    competitive binding assays, such as radioimmunoassays (RIA), enzyme    immunoassays (EIA) and sandwich competition assays, and the    different variants thereof known per se in the art; as well as the    other techniques mentioned herein. As will be clear to the skilled    person, and as described on pages 53-56 of WO 08/020,079, the    dissociation constant may be the actual or apparent dissociation    constant. Methods for determining the dissociation constant will be    clear to the skilled person, and for example include the techniques    mentioned on pages 53-56 of WO 08/020,079.-   p) The half-life of an amino acid sequence, compound or polypeptide    of the invention can generally be defined as described in    paragraph o) on page 57 of WO 08/020,079 and as mentioned therein    refers to the time taken for the serum concentration of the amino    acid sequence, compound or polypeptide to be reduced by 50%, in    vivo, for example due to degradation of the sequence or compound    and/or clearance or sequestration of the sequence or compound by    natural mechanisms. The in vivo half-life of an amino acid sequence,    compound or polypeptide of the invention can be determined in any    manner known per se, such as by pharmacokinetic analysis. Suitable    techniques will be clear to the person skilled in the art, and may    for example generally be as described in paragraph o) on page 57 of    WO 08/020,079. As also mentioned in paragraph o) on page 57 of WO    08/020,079, the half-life can be expressed using parameters such as    the t1/2-alpha, t1/2-beta and the area under the curve (AUC).    Reference is for example made to the Experimental Part below, as    well as to the standard handbooks, such as Kenneth, A et al:    Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists    and Peters et al, Pharmacokinete analysis: A Practical Approach    (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi & D    Perron, published by Marcel Dekker, 2nd Rev. edition (1982). The    terms “increase in half-life” or “increased half-life” as also as    defined in paragraph o) on page 57 of WO 08/020,079 and in    particular refer to an increase in the t1/2-beta, either with or    without an increase in the t1/2-alpha and/or the AUC or both.-   q) In the context of the present invention, “modulating” or “to    modulate” generally means either reducing or inhibiting the activity    of, or alternatively increasing the activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay. In particular, “modulating” or “to modulate” may mean either    reducing or inhibiting the activity of, or alternatively increasing    a (relevant or intended) biological activity of, a target or    antigen, as measured using a suitable in vitro, cellular or in vivo    assay (which will usually depend on the target or antigen involved),    by at least 1%, preferably at least 5%, such as at least 10% or at    least 25%, for example by at least 50%, at least 60%, at least 70%,    at least 80%, or 90% or more, compared to activity of the target or    antigen in the same assay under the same conditions but without the    presence of the construct of the invention.

As will be clear to the skilled person, “modulating” may also involveeffecting a change (which may either be an increase or a decrease) inaffinity, avidity, specificity and/or selectivity of a target or antigenfor one or more of its ligands, binding partners, partners forassociation into a homomultimeric or heteromultimeric form, orsubstrates; and/or effecting a change (which may either be an increaseor a decrease) in the sensitivity of the target or antigen for one ormore conditions in the medium or surroundings in which the target orantigen is present (such as pH, ion strength, the presence ofco-factors, etc.), compared to the same conditions but without thepresence of the construct of the invention. As will be clear to theskilled person, this may again be determined in any suitable mannerand/or using any suitable assay known per se, depending on the target orantigen involved.

“Modulating” may also mean effecting a change (i.e. an activity as anagonist, as an antagonist or as a reverse agonist, respectively,depending on the target or antigen and the desired biological orphysiological effect) with respect to one or more biological orphysiological mechanisms, effects, responses, functions, pathways oractivities in which the target or antigen (or in which its substrate(s),ligand(s) or pathway(s) are involved, such as its signalling pathway ormetabolic pathway and their associated biological or physiologicaleffects) is involved. Again, as will be clear to the skilled person,such an action as an agonist or an antagonist may be determined in anysuitable manner and/or using any suitable (in vitro and usually cellularor in assay) assay known per se, depending on the target or antigeninvolved. In particular, an action as an agonist or antagonist may besuch that an intended biological or physiological activity is increasedor decreased, respectively, by at least 1%, preferably at least 5%, suchas at least 10% or at least 25%, for example by at least 50%, at least60%, at least 70%, at least 80%, or 90% or more, compared to thebiological or physiological activity in the same assay under the sameconditions but without the presence of the construct of the invention.

Modulating may for example also involve allosteric modulation of thetarget or antigen; and/or reducing or inhibiting the binding of thetarget or antigen to one of its substrates or ligands and/or competingwith a natural ligand, substrate for binding to the target or antigen.Modulating may also involve activating the target or antigen or themechanism or pathway in which it is involved. Modulating may for examplealso involve effecting a change in respect of the folding orconfirmation of the target or antigen, or in respect of the ability ofthe target or antigen to fold, to change its confirmation (for example,upon binding of a ligand), to associate with other (sub)units, or todisassociate.

Modulating may for example also involve effecting a change in theability of the target or antigen to transport other compounds or toserve as a channel for other compounds (such as ions).

Modulating may be reversible or irreversible, but for pharmaceutical andpharmacological purposes will usually be in a reversible manner.

-   r) In respect of a target or antigen, the term “interaction site” on    the target or antigen means a site, epitope, antigenic determinant,    part, domain or stretch of amino acid residues on the target or    antigen that is a site for binding to a ligand, receptor or other    binding partner, a catalytic site, a cleavage site, a site for    allosteric interaction, a site involved in multimerisation (such as    homomerization or heterodimerization and is in particular    heterodimerization) of the target or antigen; or any other site,    epitope, antigenic determinant, part, domain or stretch of amino    acid residues on the target or antigen that is involved in a    biological action or mechanism of the target or antigen. More    generally, an “interaction site” can be any site, epitope, antigenic    determinant, part, domain or stretch of amino acid residues on the    target or antigen to which an amino acid sequence or polypeptide of    the invention can bind such that the target or antigen (and/or any    pathway, interaction, signalling, biological mechanism or biological    effect in which the target or antigen is involved) is modulated (as    defined herein).-   s) An amino acid sequence or polypeptide is said to be “specific    for” a first target or antigen compared to a second target or    antigen when is binds to the first antigen with an affinity (as    described above, and suitably expressed as a K_(D) value, K_(A)    value, K_(off) rate and/or K_(on) rate) that is at least 10 times,    such as at least 100 times, and preferably at least 1000 times, and    up to 10.000 times or more better than the affinity with which said    amino acid sequence or polypeptide binds to the second target or    polypeptide. For example, the first antigen may bind to the target    or antigen with a K_(D) value that is at least 10 times less, such    as at least 100 times less, and preferably at least 1000 times less,    such as 10.000 times less or even less than that, than the K_(D)    with which said amino acid sequence or polypeptide binds to the    second target or polypeptide. Preferably, when an amino acid    sequence or polypeptide is “specific for” a first target or antigen    compared to a second target or antigen, it is directed against (as    defined herein) said first target or antigen, but not directed    against said second target or antigen.-   t) The terms “cross-block”, “cross-blocked” and “cross-blocking” are    used interchangeably herein to mean the ability of an immunoglobulin    single variable domain or polypeptide to interfere with the binding    directly or indirectly through allosteric modulation of other    immunoglobulin single variable domains or polypeptides of the    invention to a given target. The extend to which an immunoglobulin    single variable domain or polypeptide of the invention is able to    interfere with the binding of another to the target, and therefore    whether it can be said to cross-block according to the invention,    can be determined using competition binding assays. One particularly    suitable quantitative cross-blocking assay uses a FACS- or an    AlphaScreen-based approach to measure competition between the    labelled (e.g. His tagged, biotinylated or radioactive labelled)    immunoglobulin single variable domain or polypeptide according to    the invention and the other binding agent in terms of their binding    to the target. The experimental part generally describes suitable    FACS-based assays for determining whether a binding molecule    cross-blocks or is capable of cross-blocking an immunoglobulin    single variable domain or polypeptide according to the invention. It    will be appreciated that the assay can be used with any of the    immunoglobulin single variable domains or other binding agents    described herein. Thus, in general, a cross-blocking amino acid    sequence or other binding agent according to the invention is for    example one which will bind to the target in the above    cross-blocking assay such that, during the assay and in the presence    of a second amino acid sequence or other binding agent of the    invention, the recorded displacement of the immunoglobulin single    variable domain or polypeptide according to the invention is up to    100% (e.g. in FACS based competition assay) of the maximum    theoretical displacement (e.g. displacement by cold (e.g. unlabeled)    immunoglobulin single variable domain or polypeptide that needs to    be cross-blocked) by the to be tested potentially cross-blocking    agent that is present in an amount of 0.4 mM or less (cross-blocking    agent may be another conventional monoclonal antibody such as IgG,    classic monovalent antibody fragments (Fab, scFv)) and/or variants    (including but not limited to wildtype or engineered diabodies,    triabodies, minibodies, VHHs, dAbs, VHs, VLs). Preferred, in a    non-limiting aspect, immunoglobulin single variable domains or    polypeptides of the invention, have a recorded displacement (as    described above) that is between 10% and 100%, more preferably    between 50% to 100%.-   u) An amino acid sequence is said to be “cross-reactive” for two    different antigens or antigenic determinants (such as serum albumin    from two different species of mammal, such as human serum albumin    and cyno serum albumin) if it is specific for (as defined herein)    both these different antigens or antigenic determinants.-   v) As further described herein, the total number of amino acid    residues in a Nanobody can be in the region of 110-130. It should    however be noted that parts, fragments, analogs or derivatives (as    further described herein) of a Nanobody are not particularly limited    as to their length and/or size, as long as such parts, fragments,    analogs or derivatives meet the further requirements outlined herein    and are also preferably suitable for the purposes described herein;-   w) As further described in paragraph q) on pages 58 and 59 of WO    08/020,079 (incorporated herein by reference), the amino acid    residues of a Nanobody are numbered according to the general    numbering for V_(H) domains given by Kabat et al. (“Sequence of    proteins of immunological interest”, US Public Health Services, NIH    Bethesda, Md., Publication No. 91), as applied to V_(HH) domains    from Camelids in the article of Riechmann and Muyldermans, J.    Immunol. Methods 2000 Jun. 23; 240 (1-2): 185-195 (see for example    FIG. 2 of this publication), and accordingly FR1 of a Nanobody    comprises the amino acid residues at positions 1-30, CDR1 of a    Nanobody comprises the amino acid residues at positions 31-35, FR2    of a Nanobody comprises the amino acids at positions 36-49, CDR2 of    a Nanobody comprises the amino acid residues at positions 50-65, FR3    of a Nanobody comprises the amino acid residues at positions 66-94,    CDR3 of a Nanobody comprises the amino acid residues at positions    95-102, and FR4 of a Nanobody comprises the amino acid residues at    positions 103-113.-   x) The Figures, Sequence Listing and the Experimental Part/Examples    are only given to further illustrate the invention and should not be    interpreted or construed as limiting the scope of the invention    and/or of the appended claims in any way, unless explicitly    indicated otherwise herein.

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, as well as to the prior art mentioned on page 59 of WO08/020,079 and to the list of references mentioned on pages 41-43 of theInternational application WO 06/040153, which prior art and referencesare incorporated herein by reference.

In accordance with the terminology used in the art (see the abovereferences), the variable domains present in naturally occurring heavychain antibodies will also be referred to as “V_(HH) domains”, in orderto distinguish them from the heavy chain variable domains that arepresent in conventional 4-chain antibodies (which will be referred tohereinbelow as “V_(H) domains”) and from the light chain variabledomains that are present in conventional 4-chain antibodies (which willbe referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have anumber of unique structural characteristics and functional propertieswhich make isolated V_(HH) domains (as well as Nanobodies based thereon,which share these structural characteristics and functional propertieswith the naturally occurring V_(HH) domains) and proteins containing thesame highly advantageous for use as functional antigen-binding domainsor proteins. In particular, and without being limited thereto, V_(HH)domains (which have been “designed” by nature to functionally bind to anantigen without the presence of, and without any interaction with, alight chain variable domain) and Nanobodies can function as a single,relatively small, functional antigen-binding structural unit, domain orprotein. This distinguishes the V_(HH) domains from the V_(H) and V_(L)domains of conventional 4-chain antibodies, which by themselves aregenerally not suited for practical application as single antigen-bindingproteins or domains, but need to be combined in some form or another toprovide a functional antigen-binding unit (as in for exampleconventional antibody fragments such as Fab fragments; in ScFv'sfragments, which consist of a V_(H) domain covalently linked to a V_(L)domain).

Because of these unique properties, the use of V_(HH) domains and ISV'sor Nanobodies as single antigen-binding proteins or as antigen-bindingdomains (i.e. as part of a larger protein or polypeptide) offers anumber of significant advantages over the use of conventional V_(H) andV_(L) domains, scFv's or conventional antibody fragments (such as Fab-or F(ab′)₂-fragments), including the advantages that are listed on pages60 and 61 of WO 08/020,079.

In a specific and preferred aspect, the invention provides ISV's orNanobodies against HER3, and in particular ISV's or Nanobodies againstHER3 from a warm-blooded animal, and more in particular ISV's orNanobodies against HER3 from a mammal, and especially ISV's orNanobodies against human HER3; as well as proteins and/or polypeptidescomprising at least one such ISV or Nanobody.

In particular, the invention provides ISV's or Nanobodies against HER3,and proteins and/or polypeptides comprising the same, that have improvedtherapeutic and/or pharmacological properties and/or other advantageousproperties (such as, for example, improved ease of preparation and/orreduced costs of goods), compared to conventional antibodies againstHER3 or fragments thereof, compared to constructs that could be based onsuch conventional antibodies or antibody fragments (such as Fab′fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and othermultispecific constructs (see for example the review by Holliger andHudson, Nat. Biotechnol. 2005 September; 23(9):1126-36)), and alsocompared to the so-called “dAb's” or similar (single) domain antibodiesthat may be derived from variable domains of conventional antibodies.These improved and advantageous properties will become clear from thefurther description herein, and for example include, without limitation,one or more of:

-   -   increased affinity and/or avidity for HER3, either in a        monovalent format, in a multivalent format (for example in a        bivalent format) and/or in a multispecific format (for example        one of the multispecific formats described herein below);    -   better suitability for formatting in a multivalent format (for        example in a bivalent format);    -   better suitability for formatting in a multispecific format (for        example one of the multispecific formats described herein        below);    -   improved suitability or susceptibility for “humanizing”        substitutions (as defined herein);    -   less immunogenicity, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described herein below);    -   increased stability, either in a monovalent format, in a        multivalent format (for example in a bivalent format) and/or in        a multispecific format (for example one of the multispecific        formats described herein below);    -   increased specificity towards HER3, either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described herein below);    -   decreased or where desired increased cross-reactivity with HER3        from different species;        and/or    -   one or more other improved properties desirable for        pharmaceutical use (including prophylactic use and/or        therapeutic use) and/or for diagnostic use (including but not        limited to use for imaging purposes), either in a monovalent        format, in a multivalent format (for example in a bivalent        format) and/or in a multispecific format (for example one of the        multispecific formats described herein below).

As generally described herein for the amino acid sequences of theinvention, the ISV's or Nanobodies of the invention are preferably inessentially isolated form (as defined herein), or form part of a proteinor polypeptide of the invention (as defined herein), which may compriseor essentially consist of one or more ISV's or Nanobodies of theinvention and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or more aminoacid sequences of the invention may be used as a binding unit in such aprotein or polypeptide, which may optionally contain one or more furtheramino acid sequences that can serve as a binding unit (i.e. against oneor more other targets than HER3), so as to provide a monovalent,multivalent or multispecific polypeptide of the invention, respectively,all as described herein. In particular, such a protein or polypeptidemay comprise or essentially consist of one or more ISV's or Nanobodiesof the invention and optionally one or more (other) ISV's or Nanobodies(i.e. directed against other targets than HER3), all optionally linkedvia one or more suitable linkers, so as to provide a monovalent,multivalent or multispecific ISV or Nanobody construct, respectively, asfurther described herein. Such proteins or polypeptides may also be inessentially isolated form (as defined herein).

In an ISV or Nanobody of the invention, the binding site for bindingagainst HER3 is preferably formed by the CDR sequences. Optionally, anISV or Nanobody of the invention may also, and in addition to the atleast one binding site for binding against HER3, contain one or morefurther binding sites for binding against other antigens, proteins ortargets. For methods and positions for introducing such second bindingsites, reference is for example made to Keck and Huston, BiophysicalJournal, 71, October 1996, 2002-2011; EP 0 640 130; and WO 06/07260.

As generally described herein for the amino acid sequences of theinvention, when an ISV or Nanobody of the invention (or a polypeptide ofthe invention comprising the same) is intended for administration to asubject (for example for therapeutic and/or diagnostic purposes asdescribed herein), it is preferably directed against human HER3; whereasfor veterinary purposes, it is preferably directed against HER3 from thespecies to be treated. Also, as with the amino acid sequences of theinvention, an ISV or Nanobody of the invention may or may not becross-reactive (i.e. directed against HER3 from two or more species ofmammal, such as against human HER3 and HER3 from at least one of thespecies of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequencesof the invention, the ISV's or Nanobodies of the invention may generallybe directed against any antigenic determinant, epitope, part, domain,subunit or confirmation (where applicable) of HER3. However, it isgenerally assumed and preferred that the ISV's or Nanobodies of theinvention (and polypeptides comprising the same) are directed againstthe Heregulin (or “HRG”) binding site and the heterodimerizationinteraction site.

As already described herein, the amino acid sequence and structure of anISV or Nanobody can be considered—without however being limitedthereto—to be comprised of four framework regions or “FR's” (orsometimes also referred to as “FW's”), which are referred to in the artand herein as “Framework region 1” or “FR1”; as “Framework region 2” or“FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or“FR4”, respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively. Somepreferred framework sequences and CDR's (and combinations thereof) thatare present in the ISV's or Nanobodies of the invention are as describedherein. Other suitable CDR sequences can be obtained by the methodsdescribed herein.

According to a non-limiting but preferred aspect of the invention, (theCDR sequences present in) the ISV's or Nanobodies of the invention aresuch that:

-   -   the ISV's or Nanobodies can bind to HER3 with a dissociation        constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less, and        preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably        10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association constant        (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷        to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²        liter/moles);        and/or such that:    -   the ISV's or Nanobodies can bind to HER3 with a k_(on)-rate of        between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and        10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   the ISV's or Nanobodies can bind to HER3 with a k_(off) rate        between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a near        irreversible complex with a t_(1/2) of multiple days),        preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably        between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶        s⁻¹.

Preferably, (the CDR sequences present in) the ISV's or Nanobodies ofthe invention are such that: a monovalent ISV or Nanobody of theinvention (or a polypeptide that contains only one ISV or Nanobody ofthe invention) is preferably such that it will bind to HER3 with anaffinity less than 500 nM, preferably less than 100 nM, more preferablyless than 10 nM, such as less than 5 nM.

The affinity of the ISV or Nanobody of the invention against HER3 can bedetermined in a manner known per se, for example using the generaltechniques for measuring K_(D). K_(A), k_(off) or k_(on) mentionedherein, as well as some of the specific assays described herein.

Some preferred IC50 values for binding of the ISV's or Nanobodies of theinvention (and of polypeptides comprising the same) to HER3 will becomeclear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to an ISVor Nanobody (as defined herein) against HER3, which consists of 4framework regions (FR1 to FR4 respectively) and 3 complementaritydetermining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;    and/or    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;    and/or    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;    or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, theinvention relates to an ISV or Nanobody (as defined herein) againstHER3, which consists of 4 framework regions (FR1 to FR4 respectively)and 3 complementarity determining regions (CDR1 to CDR3 respectively),in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 57 to 71;

-   b) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 57 to    71;    and    -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 87 to 101;

-   e) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 87 to    101;    and    -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 117 to 131;

-   h) amino acid sequences that have at least 80% amino acid identity    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference    with at least one of the amino acid sequences of SEQ ID NO's: 117 to    131;    or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of theinvention, when an ISV or Nanobody of the invention contains one or moreCDR1 sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)    and/or c) is preferably, and compared to the corresponding CDR    according to a), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to b) and/or c) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to a);    and/or-   iii) the CDR according to b) and/or c) may be a CDR that is derived    from a CDR according to a) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Similarly, when an ISV or Nanobody of the invention contains one or moreCDR2 sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)    and/or f) is preferably, and compared to the corresponding CDR    according to d), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to e) and/or f), preferably only contains    amino acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to d);    and/or-   iii) the CDR according to e) and/or f) may be a CDR that is derived    from a CDR according to d) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

Also, similarly, when an ISV or Nanobody of the invention contains oneor more CDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)    and/or i) is preferably, and compared to the corresponding CDR    according to g), a conservative amino acid substitution (as defined    herein);    and/or-   ii) the CDR according to h) and/or i) preferably only contains amino    acid substitutions, and no amino acid deletions or insertions,    compared to the corresponding CDR according to g);    and/or-   iii) the CDR according to h) and/or i) may be a CDR that is derived    from a CDR according to g) by means of affinity maturation using one    or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally applyto any ISV or Nanobody of the invention that comprises one or more CDR1sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e),f), h) or i), respectively.

Of the ISV's or Nanobodies of the invention, ISV's or Nanobodiescomprising one or more of the CDR's explicitly listed above areparticularly preferred; ISV's or Nanobodies comprising two or more ofthe CDR's explicitly listed above are more particularly preferred; andISV's or Nanobodies comprising three of the CDR's explicitly listedabove are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDRsequences, as well as preferred combinations of CDR sequences andframework sequences, are mentioned in Table B-1 below, which lists theCDR sequences and framework sequences that are present in a number ofpreferred (but non-limiting) ISV's or Nanobodies of the invention. Aswill be clear to the skilled person, a combination of CDR1, CDR2 andCDR3 sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3sequences that are mentioned on the same line in Table B-1) will usuallybe preferred (although the invention in its broadest sense is notlimited thereto, and also comprises other suitable combinations of theCDR sequences mentioned in Table B-1). Also, a combination of CDRsequences and framework sequences that occur in the same clone (i.e. CDRsequences and framework sequences that are mentioned on the same line inTable B-1) will usually be preferred (although the invention in itsbroadest sense is not limited thereto, and also comprises other suitablecombinations of the CDR sequences and framework sequences mentioned inTable B-1, as well as combinations of such CDR sequences and othersuitable framework sequences, e.g. as further described herein).

Also, in the ISV's or Nanobodies of the invention that comprise thecombinations of CDR's mentioned in Table B-1, each CDR can be replacedby a CDR chosen from the group consisting of amino acid sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity (as definedherein) with the mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and    compared to the corresponding CDR sequence mentioned in Table B-1, a    conservative amino acid substitution (as defined herein);    and/or-   ii) any such CDR sequence preferably only contains amino acid    substitutions, and no amino acid deletions or insertions, compared    to the corresponding CDR sequence mentioned in Table B-1;    and/or-   iii) any such CDR sequence is a CDR that is derived by means of a    technique for affinity maturation known per se, and in particular    starting from the corresponding CDR sequence mentioned in Table B-1.

However, as will be clear to the skilled person, the (combinations of)CDR sequences, as well as (the combinations of) CDR sequences andframework sequences mentioned in Table B-1 will generally be preferred.

TABLE B-1 Preferred combinations of CDR sequences, preferredcombinations of framework sequences, and preferred combinations offramework and CDR sequences. (“ID” refers to the SEQ ID NO as usedherein) Clone ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 18F0542 EVQLVESGGG 57 SYWMY 72 WVRQAPG  87 AISPGGVE 102 RFTISRDNAKNT 117LTSFATP 132 ESQGTQV LVQPGGSLRL KGVEWVS RYTDSVKG LYLQMNSLKSE TVSSSCVASGFTFS DTAMYYCAR 17B05 43 EVQLVESGGG 58 LNAMA 73 WYRQAPG  88GIFGVGST 103 RFTISRDIAKNTV 118 SSVTRGSSDY 133 WGQGTQ LVQPGGSLRL KERELVARYADSVKG FLQMNSLNSEDT VTVSS SCAASGSIGG AVYYCRM 18B05 44 EVQLVESGGG 59SAPMG 74 WYRQAPG  89 YISGDERI 104 RFTISRDTTKNT 119 DVKVRH 134 WGQGTQLVQAGGSLRL KERELVA WYGDSVKG LYLQMNSLKPE VTVSS SCAASGLTFG DTAVYYCVS 04C0745 EVQLVESGGG 60 SYPMS 75 WVRQAPG  90 TVSPGGITT 105 RFTISRDNAKNT 120DLNN 135 RGQGTQV LVQAGGSLRL KGPAWVS SYADSVKG LYLQMNSLKPE TVSS SCAASGFTFSDTAVYYCLR 18G11 46 EVQLVESGGG 61 INAMG 76 WYRQAPG  91 LITSSDTTD 106RFTISRDNTWNA 121 DHYSMGVPEKRVIM 136 YGQGTQV LVQPGGSLRL KRRELVA YAESVEGVYLQMNSLKPE TVSS SCAASGTLFK DTAVYYCHS 18E08 47 EVQLVESGGG 62 INAMG 77WYRQAPG  92 LITSSDTTD 107 RFTISRDNTWNA 122 DHYSMGVPEKRVIM 137 YGQGTQVLVQPGGSLRL KQRELVA YAESVEG VYLQMNSLKPE TVSS SCAASGTLFK DTAVYYCHS 34C0748 EVQLVESGGG 63 INAMA 78 WYRQAPG  93 EITAGGST 108 RFTISVDNAWNT 123DHYTTWDRRSAY 138 WGQGTQ LVQPGGSLGL KQRELVA NYADSVKG LYLQMNSLKVE VTVSSSCVASGSIFR DTAVYYCNL 05A09 49 EVQLVESGGG 64 DYAIG 79 WFRQAPG  94CISSSDGST 109 RFTISSDNAKNT 124 ERRRGYSDLCRFYY 139 WGKGTQ LVQAGGSLRLKEREGVS VYADSVKG VYLQMNSLKPE GMDY VTVSS SCAASGFTFD DTAVYYCAA 17C08 50EVQLVESGGG 65 SYALG 80 WFRRAPG  95 ATDRLGD 110 RFTISRDNAKNT 125GAVRYGVSTSPMN 140 WGQGTQ LMQAGDSLR KERECVA NTYFPDSV LYLQMNNLKPE YNYVTVSS LSCAASGRAFS KG DTAVYYCAA 21B02 51 EVQLVESGGG 66 YYTIG 81 WFRQAPG 96 CISSRDGD 111 RFTISRDNAKNT 126 SASDYGLGLELFHD 141 WGQGTQ LVQPGGSLRLKEREGVS SYYADSVKG AYLQMNSLKPE EYNY VTVSS SCAASGFTFD DTAVYYCAA 21F06 52EVQLVESGGG 67 LNAMG 82 WFRQGPG  97 AIDWSDGN 112 RFTISRDNAKNT 127DTPPWGPMIYIESYDS 142 WGQGTQ LVQAGGSLRL KDREFVA KDYADSV VYLQMNSLKPE VTVSSSCAASGRTYY KG DTAVYYCAA 23F05 53 EVQLVESGGG 68 GYAIG 83 WFRQAPG  98CISGGDGR 113 RFTVSSDNAKNT 128 IWGPYCSDSYEYLY 143 WGQGTQ LVQAGGSLRLKEREGVS SYYADSVKG LYLEMNSLKPED EYDY VTVSS SCAASGFTFD TAVYYCAV 34A04 54EVQLVESGGG 69 DYTIG 84 WFRQAPG  99 CISNNDGS 114 RFTISSDNAKNT 129SPHGCWYDLIPLQA 144 WGQGTQ LVQAGGSLRL KEREEIS TYYTNSVKG VYLQMNSLKPE DFGSVTVSS SCAASGFTFD DTAVYYCAA 17E08 55 EVQLVESGGG 70 LNAMG 85 WYRQTPG 100GITSITRVG 115 RFTISGDYAKNT 130 SIVKSGGADY 145 WGQGTQ LVQPGGSLRL KERELVASTRYADSA VYLQMNSLKPE VTVSS SCSASGSIFG KG DTGVYYCRM 4F10 56 EVQLVESGGG 71FYHMA 86 WYRQAPG 101 RIYTGGDTI 116 RFTISRDNSKNT 131 FREYHI 146 WGQGTQLVQPGGSLKL EQRELVA YGDSVLG VYLQMNTLKPE VTVSS SCVASGSMFR DTGVYYCNA

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2and CDR3 sequences present is suitably chosen from the group consistingof the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1;or from the group of CDR1, CDR2 and CDR3 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% “sequence identity” (as definedherein) with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” (as defined herein) with at least one ofthe CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

In this context, by “suitably chosen” is meant that, as applicable, aCDR1 sequence is chosen from suitable CDR1 sequences (i.e. as definedherein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. asdefined herein), and a CDR3 sequence is chosen from suitable CDR3sequence (i.e. as defined herein), respectively. More in particular, theCDR sequences are preferably chosen such that the Nanobodies of theinvention bind to HER3 with an affinity (suitably measured and/orexpressed as a K_(D)-value (actual or apparent), a K_(A)-value (actualor apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively asan IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1; and/or from thegroup consisting of the CDR3 sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the CDR3 sequences listed inTable B-1.

Preferably, in the Nanobodies of the invention, at least two of theCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 “amino acid difference(s)” with at least one of the CDR1, CDR2 andCDR3 sequences, respectively, listed in Table B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 sequences listed in Table B-1 or from the group of CDR3 sequencesthat have at least 80%, preferably at least 90%, more preferably atleast 95%, even more preferably at least 99% sequence identity with atleast one of the CDR3 sequences listed in Table B-1, respectively; andat least one of the CDR1 and CDR2 sequences present is suitably chosenfrom the group consisting of the CDR1 and CDR2 sequences, respectively,listed in Table B-1 or from the group of CDR1 and CDR2 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1 and CDR2 sequences, respectively,listed in Table B-1; and/or from the group consisting of the CDR1 andCDR2 sequences, respectively, that have 3, 2 or only 1 amino aciddifference(s) with at least one of the CDR1 and CDR2 sequences,respectively, listed in Table B-1.

Most preferably, in the Nanobodies of the invention, all three CDR1,CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1 or from the group of CDR1, CDR2 and CDR3 sequences,respectively, that have at least 80%, preferably at least 90%, morepreferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the CDR1, CDR2 and CDR3 sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at least oneof the CDR1, CDR2 and CDR3 sequences present is suitably chosen from thegroup consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, at least one orpreferably both of the other two CDR sequences present are suitablychosen from CDR sequences that have at least 80%, preferably at least90%, more preferably at least 95%, even more preferably at least 99%sequence identity with at least one of the corresponding CDR sequences,respectively, listed in Table B-1; and/or from the group consisting ofthe CDR sequences that have 3, 2 or only 1 amino acid difference(s) withat least one of the corresponding sequences, respectively, listed inTable B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence present is suitably chosen from the group consisting of theCDR3 listed in Table B-1. Preferably, in this aspect, at least one andpreferably both of the CDR1 and CDR2 sequences present are suitablychosen from the groups of CDR1 and CDR2 sequences, respectively, thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with the CDR1and CDR2 sequences, respectively, listed in Table B-1; and/or from thegroup consisting of the CDR1 and CDR2 sequences, respectively, that have3, 2 or only 1 amino acid difference(s) with at least one of the CDR1and CDR2 sequences, respectively, listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, at least twoof the CDR1, CDR2 and CDR3 sequences present are suitably chosen fromthe group consisting of the CDR1, CDR2 and CDR3 sequences, respectively,listed in Table B-1. Preferably, in this aspect, the remaining CDRsequence present is suitably chosen from the group of CDR sequences thathave at least 80%, preferably at least 90%, more preferably at least95%, even more preferably at least 99% sequence identity with at leastone of the corresponding CDR sequences listed in Table B-1; and/or fromthe group consisting of CDR sequences that have 3, 2 or only 1 aminoacid difference(s) with at least one of the corresponding sequenceslisted in Table B-1.

In particular, in the Nanobodies of the invention, at least the CDR3sequence is suitably chosen from the group consisting of the CDR3sequences listed in Table B-1, and either the CDR1 sequence or the CDR2sequence is suitably chosen from the group consisting of the CDR1 andCDR2 sequences, respectively, listed in Table B-1. Preferably, in thisaspect, the remaining CDR sequence present is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with at least one of the corresponding CDR sequences listed inTable B-1; and/or from the group consisting of CDR sequences that have3, 2 or only 1 amino acid difference(s) with the corresponding CDRsequences listed in Table B-1.

Even more preferably, in the Nanobodies of the invention, all threeCDR1, CDR2 and CDR3 sequences present are suitably chosen from the groupconsisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed inTable B-1.

Also, generally, the combinations of CDR's listed in Table B-1 (i.e.those mentioned on the same line in Table B-1) are preferred. Thus, itis generally preferred that, when a CDR in a Nanobody of the inventionis a CDR sequence mentioned in Table B-1 or is suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with a CDR sequence listed in Table B-1; and/or from the groupconsisting of CDR sequences that have 3, 2 or only 1 amino aciddifference(s) with a CDR sequence listed in Table B-1, that at least oneand preferably both of the other CDR's are suitably chosen from the CDRsequences that belong to the same combination in Table B-1 (i.e.mentioned on the same line in Table B-1) or are suitably chosen from thegroup of CDR sequences that have at least 80%, preferably at least 90%,more preferably at least 95%, even more preferably at least 99% sequenceidentity with the CDR sequence(s) belonging to the same combinationand/or from the group consisting of CDR sequences that have 3, 2 or only1 amino acid difference(s) with the CDR sequence(s) belonging to thesame combination. The other preferences indicated in the aboveparagraphs also apply to the combinations of CDR's mentioned in TableB-1.

Thus, by means of non-limiting examples, a Nanobody of the invention canfor example comprise a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1, a CDR2sequence that has 3, 2 or 1 amino acid difference with one of the CDR2sequences mentioned in Table B-1 (but belonging to a differentcombination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1)a CDR1 sequence that has more than 80% sequence identity with one of theCDR1 sequences mentioned in Table B-1; a CDR2 sequence that has 3, 2 or1 amino acid difference with one of the CDR2 sequences mentioned inTable B-1 (but belonging to a different combination); and a CDR3sequence that has more than 80% sequence identity with one of the CDR3sequences mentioned in Table B-1 (but belonging to a differentcombination); or (2) a CDR1 sequence that has more than 80% sequenceidentity with one of the CDR1 sequences mentioned in Table B-1; a CDR2sequence, and one of the CDR3 sequences listed in Table B-1; or (3) aCDR1 sequence; a CDR2 sequence that has more than 80% sequence identitywith one of the CDR2 sequence listed in Table B-1; and a CDR3 sequencethat has 3, 2 or 1 amino acid differences with the CDR3 sequencementioned in Table B-1 that belongs to the same combination as the CDR2sequence.

Some particularly preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; a CDR2 sequencethat has 3, 2 or 1 amino acid difference with the CDR2 sequencementioned in Table B-1 that belongs to the same combination; and a CDR3sequence that has more than 80% sequence identity with the CDR3 sequencementioned in Table B-1 that belongs to the same combination; (2) a CDR1sequence; a CDR 2 listed in Table B-1 and a CDR3 sequence listed inTable B-1 (in which the CDR2 sequence and CDR3 sequence may belong todifferent combinations).

Some even more preferred Nanobodies of the invention may for examplecomprise: (1) a CDR1 sequence that has more than 80% sequence identitywith one of the CDR1 sequences mentioned in Table B-1; the CDR2 sequencelisted in Table B-1 that belongs to the same combination; and a CDR3sequence mentioned in Table B-1 that belongs to a different combination;or (2) a CDR1 sequence mentioned in Table B-1; a CDR2 sequence that has3, 2 or 1 amino acid differences with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and a CDR3 sequence thathas more than 80% sequence identity with the CDR3 sequence listed inTable B-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for examplecomprise a CDR1 sequence mentioned in Table B-1, a CDR2 sequence thathas more than 80% sequence identity with the CDR2 sequence mentioned inTable B-1 that belongs to the same combination; and the CDR3 sequencementioned in Table B-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 andCDR3 sequences present are suitably chosen from one of the combinationsof CDR1, CDR2 and CDR3 sequences, respectively, listed in Table B-1.

According to another preferred, but non-limiting aspect of the invention(a) CDR1 has a length of between 1 and 12 amino acid residues, andusually between 2 and 9 amino acid residues, such as 5, 6 or 7 aminoacid residues; and/or (b) CDR2 has a length of between 13 and 24 aminoacid residues, and usually between 15 and 21 amino acid residues, suchas 16 and 17 amino acid residues; and/or (c) CDR3 has a length ofbetween 2 and 35 amino acid residues, and usually between 3 and 30 aminoacid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences (as defined herein) have more than80%, preferably more than 90%, more preferably more than 95%, such as99% or more sequence identity (as defined herein) with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 12 to 26(see Table A-1).

Generally, Nanobodies with the above CDR sequences may be as furtherdescribed herein, and preferably have framework sequences that are alsoas further described herein.

For example, as already mentioned, the framework sequences present inthe nanobodies may be as generally described on pages 258 to 297 of WO09/068,627 (incorporated herein by reference). For example, they maycontain one or more of the combinations of Hallmark residues set out inTable A-5 of WO 09/068,627; and FR1, FR2, FR3 and FR4 may contain theamino acid residues set out in Table A-6, Table A-7, Table A-8 and TableA-9 of WO 09/068,627, respectively. Also, when the ISV's of theinvention are Nanobodies, they may belong to the KERE-group (see pages281 to 284 of WO 09/068,627, with some representative FR1, FR2, FR3 andFR4 sequences for this group given in Tables A-11/A-15, A-12, A-13 andA-14 of WO 09/068,627); to the GLEW-group (see pages 285 to 287 of WO09/068,627, with some representative FR1, FR2, FR3 and FR4 sequences forthis group given in Tables A-16/A-20, A-17, A-18 and A-19 of WO09/068,627); or to the P, R, S 103 group (see pages 287 to 291 of WO09/068,627, with some representative FR1, FR2, FR3 and FR4 sequences forthis group given in Tables A-21/A-25, A-22, A-23 and A-24 of WO09/068,627), which are all as described in WO 09/068,627, with somerepresentative sequences for each of these groups given in Table A-10 ofWO 09/068,627. As also described in WO 09/068,627, these frameworksequences may contain one or more suitable humanizing substitutions or(other) substitutions for optimizing the sequence (see also the furtherdisclosure herein).

Again, some particularly preferred but non-limiting FR1, FR2, FR3 andFR4 sequences (and combinations thereof) are those described in TableB-1, or suitable variants of such FR1, FR2, FR3 and FR4 sequences,respectively (for example, with less than 5, such as 1, 2, 3, 4 or 5suitable amino acid differences in such an FR1, FR2, FR3 or FR4 comparedto a framework sequence mentioned in Table B-1, in which the amino aciddifferences may be as described in WO 09/068,627) that still essentiallyretain the desired properties of Nanobodies.

Thus, for example and as mentioned herein, such Nanobodies may benaturally occurring Nanobodies (from any suitable species), naturallyoccurring V_(HH) sequences (i.e. from a suitable species of Camelid) orsynthetic or semi-synthetic amino acid sequences or Nanobodies,including but not limited to partially humanized Nanobodies or V_(HH)sequences, fully humanized Nanobodies or V_(HH) sequences, camelizedheavy chain variable domain sequences, as well as Nanobodies that havebeen obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates toa humanized Nanobody, which consists of 4 framework regions (FR1 to FR4respectively) and 3 complementarity determining regions (CDR1 to CDR3respectively), in which CDR1 to CDR3 are as defined herein and in whichsaid humanized Nanobody comprises at least one humanizing substitution(as defined herein), and in particular at least one humanizingsubstitution in at least one of its framework sequences (as definedherein).

Also, in addition to humanizing substitutions as described herein, theISV's and in particular nanobodies of the invention may contain one ormore other/further substitutions. Again, some preferred, butnon-limiting examples of such other/further substitutions will becomeclear from the further description herein, and for example may include(and preferably essentially consist of) one or more of the followingsubstitutions:

-   (a) one or more conservative amino acid substitutions; and/or-   (b) one or more substitutions in which a “camelid” amino acid    residue at a certain position is replaced by a different “camelid”    amino acid residue that occurs at said position, for which reference    is for example made to Tables A-6 to A-9 from PCT/EP2008/066365    (published on Jun. 4, 2009 as WO 09/068,627), which mention the    various Camelid residues that occur as each amino acid position in    wild-type VHH's. Such substitutions may even comprise suitable    substitutions of an amino acid residue that occurs at a Hallmark    position with another amino acid residue that occurring at a    Hallmark position in a wild-type VHH (for which reference is for    example made to Tables A-6 to A-9 from PCT/EP2008/066365); and/or-   (c) one or more substitutions that improve the (other) properties of    the protein, such as substitutions that improve the long-term    stability and/or properties under storage of the protein. These may    for example and without limitation be substitutions that prevent or    reduce oxidation events (for example, of methionine residues); that    prevent or reduce pyroglutamate formation; and/or that prevent or    reduce isomerisation or deamidation of aspartic acids or asparagines    (for example, of DG, DS, NG or NS motifs). For such substitutions,    reference is for example made to the International application WO    09/095,235, which is generally directed to methods for stabilizing    single immunoglobulin variable domains by means of such    substitutions, and also gives some specific example of suitable    substitutions (see for example pages 4 and 5 and pages 10 to 15).    One example of such substitution may be to replace an NS motif at    positions 82a and 82b with an NN motif.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody in which the CDR sequences have at least 70% amino acididentity, preferably at least 80% amino acid identity, more preferablyat least 90% amino acid identity, such as 95% amino acid identity ormore or even essentially 100% amino acid identity with the CDR sequencesof at least one of the amino acid sequences of SEQ ID NO's: 12 to 26(see Table A-1). This degree of amino acid identity can for example bedetermined by determining the degree of amino acid identity (in a mannerdescribed herein) between said Nanobody and one or more of the sequencesof SEQ ID NO's: 12 to 26 (see Table A-1), in which the amino acidresidues that form the framework regions are disregarded. SuchNanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates toa Nanobody with an amino acid sequence that is chosen from the groupconsisting of SEQ ID NO's: 12 to 26 (see Table A-1) or from the groupconsisting of from amino acid sequences that have more than 80%,preferably more than 90%, more preferably more than 95%, such as 99% ormore sequence identity (as defined herein) with at least one of theamino acid sequences of SEQ ID NO's: 12 to 26 (see Table A-1).

Another preferred, but non-limiting aspect of the invention relates tohumanized variants of the Nanobodies of SEQ ID NO's: 12 to 26 (see TableA-1), that comprise, compared to the corresponding native V_(HH)sequence, at least one humanizing substitution (as defined herein), andin particular at least one humanizing substitution in at least one ofits framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of atleast one Nanobody of the invention. Some preferred, but non-limitingexamples of polypeptides of the invention are given in SEQ ID NO's: 147to 327, more preferably HER3MS00135 (SEQ ID NO:282), HER3MS00212 (SEQ IDNO:319) or HER3MS00215 (SEQ ID NO:322) (see Table A-2). It should benoted that some of the sequences listed in the Table below (SEQ ID NO's:224-231, 241-281, 318 and SEQ ID NO's: 323-327) contain a C-terminal tag(e.g. a His-tag of 6H). In practice, these polypeptides may also be used(and for example for therapeutic purposes preferably are used) withoutthe (C-terminal) tag, and the (C-terminal) tag should also bedisregarded for the purposes of determining the degree of sequenceidentity to each of these sequences.

TABLE A-2 Preferred polypeptide or compound sequences (also referredherein as a sequence with a particular name or SEQ ID NO: X, wherein Xis a number referring to the relevant amino acid sequence): SEQ ID NO:X, wherein Name X = Amino acid sequence 17C8- 147EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-REFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPE 17C8-9GS-DTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGG ALB8GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKEREFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSR SSQGTLVTVSS 17C8- 148EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-REFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPE 18F5-9GS-DTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGG ALB8GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17C8- 149EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-REFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPE 21F06-DTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGG 9GS-ALB8GGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSL SRSSQGTLVTVSS 17C8- 150EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-4C7-REFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPE 9GS-ALB8DTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F5- 151EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 17C8-9GS-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKEREFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F5- 152EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 18F5-9GS-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F5- 153EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 21F6-9GS-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F5- 154EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 34C7-9GS-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 21F6- 155EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 17C8-9GS-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKEREFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS LSRSSQGTLVTVSS 21F6- 156EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 18F5-9GS-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 21F6- 157EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 21F6-9GS-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS LSRSSQGTLVTVSS 21F6- 158EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 34C7-9GS-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS 21F6- 159EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-4C7-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 9GS-ALB8PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 34C7- 160EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 18F5-9GS-DTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 34C7- 161EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 21F6-9GS-DTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS 34C7- 162EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 34C7-9GS-DTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSG ALB8GGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 34C7- 163EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-4C7-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 9GS-ALB8DTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C7-35GS- 164EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 17C8-9GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE ALB8DTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKEREFVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C7-35GS- 165EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 21F6-9GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE ALB8DTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C7-35GS- 166EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 34C7-9GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE ALB8DTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C7-35GS- 167EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 4C7-9GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE ALB8DTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F5- 168EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 18G11-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 21F6- 169EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 18G11-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS 4C7-35GS- 170EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 18G11-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE 9GS-ALB8DTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18G11- 171EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 18F5-9GS-TAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGS ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18G11- 172EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 18G11-TAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS 18G11- 173EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 21F6-9GS-TAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGS ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS 18G11- 174EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-4C7-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 9GS-ALB8TAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F05- 175EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 4C07-9GS-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F05- 176EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 17B05-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18F05- 177EVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKG 35GS-VEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSED 17E08-TAMYYCARLTSFATPESQGTLVTVSSGGGGSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17C08- 178EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-RECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKP 18G11-EDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS 17C08- 179EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-RECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKP 34C07-EDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS 17C08- 180EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-RECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKP 17B05-EDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS 17C08- 181EVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKE 35GS-RECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKP 17E08-EDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS 4C07- 182EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 35GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE 18F05-DTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGG 9GS-ALB8SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C07- 183EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 35GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE 17B05-DTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGG 9GS-ALB8SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 4C07- 184EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKG 35GS-PAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPE 17E08-DTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGGSGGGGSGGGG 9GS-ALB8SGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18G11- 185EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 17C08-TAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKERECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS 18G11- 186EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 34C07-TAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVT VSS 18G11- 187EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 17B05-TAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 18G11- 188EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKR 35GS-RELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPED 17E08-TAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 21F06- 189EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 17B05-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS 21F06- 190EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGK 35GS-DREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLK 17E08-PEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSSGGGGSG 9GS-ALB8GGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS 34C07- 191EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 17C08-DTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKERECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS 34C07- 192EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 18G11-DTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS 34C07- 193EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 17B05-DTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 34C07- 194EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQ 35GS-RELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVE 17E08-DTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTV SS 17B05- 195EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 4C07-9GS-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17B05- 196EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 17C08-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKERECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVT VSS 17B05- 197EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 18F05-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17B05- 198EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 18G11-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17B05- 199EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 21F06-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVT VSS 17B05- 200EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 34C07-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17B05- 201EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 17B05-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17B05- 202EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKE 35GS-RELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDT 17E08-AVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGGSGGGGS 9GS-ALB8GGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 203EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 4C07-9GS-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 204EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 17C08-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLMQAGDSLRLSCAASGRAFSSYALGWFRRAPGKERECVAATDRLGDNTYFPDSVKGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCAAGAVRYGVSTSPMNYNYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGT LVTVSS 17E08- 205EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 18F05-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSYWMYWVRQAPGKGVEWVSAISPGGVERYTDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAMYYCARLTSFATPESQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 206EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 18G11-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 207EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 21F06-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS 17E08- 208EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 34C07-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 209EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 17B05-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS 17E08- 210EVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKER 35GS-ELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPE 17E08-DTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGGSGGG 9GS-ALB8GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCSASGSIFGLNAMGWYRQTPGKERELVAGITSITRVGSTRYADSAKGRFTISGDYAKNTVYLQMNSLKPEDTGVYYCRMSIVKSGGADYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00022 211EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS LSRSSQGTLVTVSS HER3MS00023212 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS HER3MS00024 213EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00026 214EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQG TLVTVSS HER3MS00028 215EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00030 216EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00031 217EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00032 218EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00034 219EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQ GTLVTVSS HER3MS00035 220EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00037 221EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS HER3MS00038 222EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSS QGTLVTVSS HER3MS00039 223EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00042 224EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLNNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHH HH HER3MS00043 225EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLNNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH HER3MS00044 226EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARDLNNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHH HH HER3MS00045 227EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARDLNNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH HER3MS00046 228EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00047 229 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00048 230 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQGPGKEREFVAAIDWSDGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQKLI SEEDLNGAAHHHHHHHER3MS00049 231 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSDGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQKLI SEEDLNGAAHHHHHHHER3MS00051 232 EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00052 233EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00054 234EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00055 235EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00056 236EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00057 237EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00058 238EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00060 239EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLV TVSS HER3MS00061 240EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVT VSS HER3MS00068 241EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSGGVPEKRVIMYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00069242 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSLGVPEKRVIMYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00070243 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSIGVPEKRVIMYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00071244 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSVGVPEKRVIMYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00072245 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVILYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00073246 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIDYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00074247 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIEYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00076248 EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVFLQMNSLRSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00077 249EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVFLQMNSLRSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00078 250EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVYLQMNSLRSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00079 251EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVFLQMNSLRSEDTAVYYCAMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00080 252EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRSEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00081 253EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVFLQMNSLRSEDTAVYYCAMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00082 254EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVYLQMNSLRSEDTAVYYCAMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00083 255EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRSEDTAVYYCAMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00084 256EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVFLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00085 257EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDISKNTVFLQMNSLRAEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS00088 258EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSYGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00089 259 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSEGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00090 260 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDANKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00091 261 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPFIYIESYDSWGQGTLVTVSSAAAEQKL ISEEDLNGAAHHHHHHHER3MS00092 262 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPYIYIESYDSWGQGTLVTVSSAAAEQKL ISEEDLNGAAHHHHHHHER3MS00093 263 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSAAAEQKL ISEEDLNGAAHHHHHHHER3MS00094 264 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYQSWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00095 265 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYESWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00096 266 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDDWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00097 267 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDEWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00098 268 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDTWGQGTLVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS00118 269 EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00119 270EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00120 271DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00121 272DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSAAAEQKLISEEDLNG AAHHHHHH HER3MS00123 273EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSAAAEQKLISEEDL NGAAHHHHHH HER3MS00124 274EVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRAEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSAAAEQKLISEEDL NGAAHHHHHH HER3MS00125 275EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKQRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00127276 DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSAAAEQKLISEEDL NGAAHHHHHH HER3MS00128 277DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRAEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSAAAEQKLISEEDL NGAAHHHHHH HER3MS00129 278EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH HER3MS00130 279EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHH HH HER3MS00131 280EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLSNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHHHH HER3MS00132 281EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLSNRGQGTLVTVSSAAAEQKLISEEDLNGAAHHHH HH HER3MS00135 282DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWG PLIYIESYDSWGQGTLVTVSSHER3MS00136 283 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSS HER3MS00137 284DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00138 285 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00139 286DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRR SAYWGQGTLVTVSS HER3MS00140287 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCN LDHYTTWDRRSAYWGQGTLVTVSSHER3MS00141 288 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG GSLSRSSQGTLVTVSSHER3MS00142 289 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPED TAVYYCTIGGSLSRSSQGTLVTVSSHER3MS00143 290 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPE KRVILYGQGTLVTVSSHER3MS00144 291 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYC HSDHYSLGVPEKRVILYGQGTLVTVSSHER3MS00145 292 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIG GSLSRSSQGTLVTVSSHER3MS00146 293 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS HER3MS00147 294EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTW DRRSAYWGQGTLVTVSSHER3MS00148 295 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTT WDRRSAYWGQGTLVTVSSHER3MS00149 296 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCT IGGSLSRSSQGTLVTVSSHER3MS00150 297 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00151 298 DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVY YCLRDLGNRGQGTLVTVSSHER3MS00152 299 DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVY YCLRDLGNRGQGTLVTVSSHER3MS00153 300 DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCT IGGSLSRSSQGTLVTVSSHER3MS00154 301 DVQLVESGGGLVQPGGSLRLSCAASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISRDNSWNTLYLQMNSLRPEDTAVYYCNLDHYTTWDRRSAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00155 302 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLG VPEKRVILYGQGTLVTVSSHER3MS00156 303 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSL GVPEKRVILYGQGTLVTVSSHER3MS00157 304 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00158 305 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00159 306 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVY YCLRDLGNRGQGTLVTVSSHER3MS00160 307 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVY YCLRDLGNRGQGTLVTVSSHER3MS00161 308 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00162 309 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY YCTIGGSLSRSSQGTLVTVSSHER3MS00199 310 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDASKNTVYLQMNSLRPEDTAVYYCAADT PPWGPLIYIESYDSWGQGTLVTVSSHER3MS00200 311 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDASKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSSHER3MS00201 312 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDASKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHY SLGVPEKRVILYGQGTLVTVSSHER3MS00202 313 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDASKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDT AVYYCTIGGSLSRSSQGTLVTVSSHER3MS00207 314 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVT RGSSDYWGQGTLVTVSSHER3MS00208 315 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYG QGTLVTVSS HER3MS00209 316DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC TIGGSLSRSSQGTLVTVSSHER3MS00210 317 EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAEDVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHY SLGVPEKRVILYGQGTLVTVSSHER3MS00211 318 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYADSVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSAAAEQKLISEED LNGAAHHHHHH HER3MS00212319 DVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVTRGSSDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYADSVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPE KRVILYGQGTLVTVSSHER3MS00213 320 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPEWVSTVSPGGITTSYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCLRDLGNRGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYADSVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYG QGTLVTVSS HER3MS00214 321EVQLVESGGGLVQPGGSLRLSCAASGRTYYLNAMGWFRQAPGKEREFVAAIDWSEGNKDYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCAADTPPWGPLIYIESYDSWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYADSVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHY SLGVPEKRVILYGQGTLVTVSSHER3MS00215 322 EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYADSVKGRFTISRDNSWNTVYLQMNSLRPEDTAVYYCHSDHYSLGVPEKRVILYGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTAVYYCRMSSVT RGSSDYWGQGTLVTVSSHER3MS004C07 323 EVQLVESGGGLVQAGGSLRLSCAASGFTFSSYPMSWVRQAPGKGPAWVSTVSPGGITTSYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCLRDLNNRGQGTQVTVSSAAAEQKLISEEDLNGAAHHH HHH HER3MS017B05 324EVQLVESGGGLVQPGGSLRLSCAASGSIGGLNAMAWYRQAPGKERELVAGIFGVGSTRYADSVKGRFTISRDIAKNTVFLQMNSLNSEDTAVYYCRMSSVTRGSSDYWGQGTQVTVSSAAAEQKLISEEDLNGA AHHHHHH HER3MS018G11 325EVQLVESGGGLVQPGGSLRLSCAASGTLFKINAMGWYRQAPGKRRELVALITSSDTTDYAESVEGRFTISRDNTWNAVYLQMNSLKPEDTAVYYCHSDHYSMGVPEKRVIMYGQGTQVTVSSAAAEQKLISEE DLNGAAHHHHHH HER3MS021F06326 EVQLVESGGGLVQAGGSLRLSCAASGRTYYLNAMGWFRQGPGKDREFVAAIDWSDGNKDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTPPWGPMIYIESYDSWGQGTQVTVSSAAAEQK LISEEDLNGAAHHHHHHHER3MS034C07 327 EVQLVESGGGLVQPGGSLGLSCVASGSIFRINAMAWYRQAPGKQRELVAEITAGGSTNYADSVKGRFTISVDNAWNTLYLQMNSLKVEDTAVYYCNLDHYTTWDRRSAYWGQGTQVTVSSAAAEQKLISEE DLNGAAHHHHHH

It will be clear to the skilled person that the Nanobodies that arementioned herein as “preferred” (or “more preferred”, “even morepreferred”, etc.) are also preferred (or more preferred, or even morepreferred, etc.) for use in the polypeptides described herein. Thus,polypeptides that comprise or essentially consist of one or more“preferred” Nanobodies of the invention will generally be preferred, andpolypeptides that comprise or essentially consist of one or more “morepreferred” Nanobodies of the invention will generally be more preferred,etc.

Generally, proteins or polypeptides that comprise or essentially consistof a single Nanobody (such as a single Nanobody of the invention) willbe referred to herein as “monovalent” proteins or polypeptides or as“monovalent constructs”. Proteins and polypeptides that comprise oressentially consist of two or more Nanobodies (such as at least twoNanobodies of the invention or at least one Nanobody of the inventionand at least one other Nanobody) will be referred to herein as“multivalent” proteins or polypeptides or as “multivalent constructs”,and these may provide certain advantages compared to the correspondingmonovalent Nanobodies of the invention. Some non-limiting examples ofsuch multivalent constructs will become clear from the furtherdescription herein.

According to one specific, but non-limiting aspect, a polypeptide of theinvention comprises or essentially consists of at least two Nanobodiesof the invention, such as two or three Nanobodies of the invention. Asfurther described herein, such multivalent constructs can providecertain advantages compared to a protein or polypeptide comprising oressentially consisting of a single Nanobody of the invention, such as amuch improved avidity for HER3. Such multivalent constructs will beclear to the skilled person based on the disclosure herein; somepreferred, but non-limiting examples of such multivalent Nanobodyconstructs are the constructs of SEQ ID NO's: 147 to 327, morepreferably HER3MS00135 (SEQ ID NO:282), HER3MS00212 (SEQ ID NO:319) orHER3MS00215 (SEQ ID NO:322). According to another specific, butnon-limiting aspect, a polypeptide of the invention comprises oressentially consists of at least one Nanobody of the invention and atleast one other binding unit (i.e. directed against another epitope,antigen, target, protein or polypeptide), which is preferably also aNanobody. Such proteins or polypeptides are also referred to herein as“multispecific” proteins or polypeptides or as ‘multispecificconstructs”, and these may provide certain advantages compared to thecorresponding monovalent Nanobodies of the invention (as will becomeclear from the further discussion herein of some preferred, but,non-limiting multispecific constructs). Such multispecific constructswill be clear to the skilled person based on the disclosure herein; somepreferred, but non-limiting examples of such multispecific Nanobodyconstructs are the constructs of SEQ ID NO's: 147 to 327, morepreferably HER3MS00135 (SEQ ID NO:282), HER3MS00212 (SEQ ID NO:319) orHER3MS00215 (SEQ ID NO:322).

According to yet another specific, but non-limiting aspect, apolypeptide of the invention comprises or essentially consists of atleast one Nanobody of the invention, optionally one or more furtherNanobodies, and at least one other amino acid sequence (such as aprotein or polypeptide) that confers at least one desired property tothe Nanobody of the invention and/or to the resulting fusion protein.Again, such fusion proteins may provide certain advantages compared tothe corresponding monovalent Nanobodies of the invention. Somenon-limiting examples of such amino acid sequences and of such fusionconstructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, forexample to provide a trivalent bispecific construct comprising twoNanobodies of the invention and one other Nanobody, and optionally oneor more other amino acid sequences. Further non-limiting examples ofsuch constructs, as well as some constructs that are particularlypreferred within the context of the present invention, will become clearfrom the further description herein.

In the above constructs, the one or more Nanobodies and/or other aminoacid sequences may be directly linked to each other and/or suitablylinked to each other via one or more linker sequences. Some suitable butnon-limiting examples of such linkers will become clear from the furtherdescription herein.

In one specific aspect of the invention, a Nanobody of the invention ora compound, construct or polypeptide of the invention comprising atleast one Nanobody of the invention may have an increased half-life,compared to the corresponding amino acid sequence of the invention. Somepreferred, but non-limiting examples of such Nanobodies, compounds andpolypeptides will become clear to the skilled person based on thefurther disclosure herein, and for example comprise Nanobodies sequencesor polypeptides of the invention that have been chemically modified toincrease the half-life thereof (for example, by means of pegylation);amino acid sequences of the invention that comprise at least oneadditional binding site for binding to a serum protein (such as serumalbumin, see for example EP 0 368 684 B1, page 4); or polypeptides ofthe invention that comprise at least one Nanobody of the invention thatis linked to at least one moiety (and in particular at least one aminoacid sequence) that increases the half-life of the Nanobody of theinvention. Examples of polypeptides of the invention that comprise suchhalf-life extending moieties or amino acid sequences will become clearto the skilled person based on the further disclosure herein; and forexample include, without limitation, polypeptides in which the one ormore Nanobodies of the invention are suitable linked to one or moreserum proteins or fragments thereof (such as serum albumin or suitablefragments thereof) or to one or more binding units that can bind toserum proteins (such as, for example, Nanobodies or (single) domainantibodies that can bind to serum proteins such as serum albumin, serumimmunoglobulins such as IgG, or transferrine); polypeptides in which aNanobody of the invention is linked to an Fc portion (such as a humanFc) or a suitable part or fragment thereof; or polypeptides in which theone or more Nanobodies of the invention are suitable linked to one ormore small proteins or peptides that can bind to serum proteins.

Again, as will be clear to the skilled person, such Nanobodies,compounds, constructs or polypeptides may contain one or more additionalgroups, residues, moieties or binding units, such as one or more furtheramino acid sequences and in particular one or more additional Nanobodies(i.e. not directed against HER3), so as to provide a tri- ofmultispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs orpolypeptides comprising the same) with increased half-life preferablyhave a half-life that is at least 1.5 times, preferably at least 2times, such as at least 5 times, for example at least 10 times or morethan 20 times, greater than the half-life of the corresponding aminoacid sequence of the invention per se. For example, the Nanobodies,compounds, constructs or polypeptides of the invention with increasedhalf-life may have a half-life that is increased with more than 1 hours,preferably more than 2 hours, more preferably more than 6 hours, such asmore than 12 hours, or even more than 24, 48 or 72 hours, compared tothe corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchNanobodies, compound, constructs or polypeptides of the inventionexhibit a serum half-life in human of at least about 12 hours,preferably at least 24 hours, more preferably at least 48 hours, evenmore preferably at least 72 hours or more. For example, compounds orpolypeptides of the invention may have a half-life of at least 5 days(such as about 5 to 10 days), preferably at least 9 days (such as about9 to 14 days), more preferably at least about 10 days (such as about 10to 15 days), or at least about 11 days (such as about 11 to 16 days),more preferably at least about 12 days (such as about 12 to 18 days ormore), or more than 14 days (such as about 14 to 19 days). Suchhalf-life extended constructs will be clear to the skilled person basedon the disclosure herein; some preferred, but non-limiting examples ofsuch multispecific Nanobody constructs are the constructs of SEQ IDNO's: 147 to 327, more preferably HER3MS00135 (SEQ ID NO:282),HER3MS00212 (SEQ ID NO:319) or HER3MS00215 (SEQ ID NO:322).

In particular, polypeptides comprising one or more Nanobodies of theinvention are preferably such that they:

-   -   bind to HER3 with a dissociation constant (K_(D)) of 10⁻⁵ to        10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²        moles/liter or less and more preferably 10⁻⁸ to 10⁻¹²        moles/liter (i.e. with an association constant (K_(A)) of 10⁵ to        10¹² liter/moles or more, and preferably 10⁷ to 10¹² liter/moles        or more and more preferably 10⁸ to 10¹² liter/moles);        and/or such that they:    -   bind to HER3 with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about        10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more        preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻s⁻¹, such as between 10⁵        M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;        and/or such that they:    -   bind to HER3 with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s)        and 10⁻⁶ s⁻¹ (providing a near irreversible complex with a        t_(1/2) of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶        s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as        between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence ofthe invention is preferably such that it will bind to HER3 with anaffinity less than 500 nM, preferably less than 200 nM, more preferablyless than 10 nM, such as less than 1 nM. In this respect, it will beclear to the skilled person that a polypeptide that contains two or moreNanobodies of the invention may bind to HER3 with an increased avidity,compared to a polypeptide that contains only one amino acid sequence ofthe invention.

Some preferred IC₅₀ values for binding of the amino acid sequences orpolypeptides of the invention to HER3 will become clear from the furtherdescription and examples herein.

Other polypeptides according to this preferred aspect of the inventionmay for example be chosen from the group consisting of amino acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the amino acid sequences of SEQ IDNO's: 147 to 327, more preferably HER3MS00135 (SEQ ID NO:282),HER3MS00212 (SEQ ID NO:319) or HER3MS00215 (SEQ ID NO:322) (see TableA-2), in which the Nanobodies comprised within said amino acid sequencesare preferably as further defined herein.

Another aspect of this invention relates to a nucleic acid that encodesan amino acid sequence of the invention (such as an ISV or Nanobody ofthe invention) or a polypeptide of the invention comprising the same.Again, as generally described herein for the nucleic acids of theinvention, such a nucleic acid may be in the form of a geneticconstruct, as defined herein.

Other nucleic acids according to a preferred aspect of the invention mayfor example be chosen from the group consisting of nucleic acidsequences that have more than 80%, preferably more than 90%, morepreferably more than 95%, such as 99% or more “sequence identity” (asdefined herein) with one or more of the nucleic acid sequences of SEQ IDNO's: 27 to 41 (see FIG. 1).

In another aspect, the invention relates to host or host cell thatexpresses or that is capable of expressing an amino acid sequence (suchas a Nanobody) of the invention and/or a polypeptide of the inventioncomprising the same; and/or that contains a nucleic acid of theinvention. Some preferred but non-limiting examples of such hosts orhost cells will become clear from the further description herein.

Another aspect of the invention relates to a product or compositioncontaining or comprising at least one amino acid sequence of theinvention, at least one polypeptide of the invention and/or at least onenucleic acid of the invention, and optionally one or more furthercomponents of such compositions known per se, i.e. depending on theintended use of the composition. Such a product or composition may forexample be a pharmaceutical composition (as described herein), aveterinary composition or a product or composition for diagnostic use(as also described herein). Some preferred but non-limiting examples ofsuch products or compositions will become clear from the furtherdescription herein.

The invention further relates to methods for preparing or generating theamino acid sequences, compounds, constructs, polypeptides, nucleicacids, host cells, products and compositions described herein. Somepreferred but non-limiting examples of such methods will become clearfrom the further description herein.

The invention further relates to applications and uses of the amino acidsequences, compounds, constructs, polypeptides, nucleic acids, hostcells, products and compositions described herein, as well as to methodsfor the prevention and/or treatment for diseases and disordersassociated with HER3. Some preferred but non-limiting applications anduses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the inventionwill also become clear from the further description herein below.

Generally, it should be noted that the term Nanobody as used herein inits broadest sense is not limited to a specific biological source or toa specific method of preparation. For example, as will be discussed inmore detail below, the Nanobodies of the invention can generally beobtained by any of the techniques (1) to (8) mentioned on pages 61 and62 of WO 08/020,079, or any other suitable technique known per se. Onepreferred class of Nanobodies corresponds to the V_(HH) domains ofnaturally occurring heavy chain antibodies directed against HER3. Asfurther described herein, such V_(HH) sequences can generally begenerated or obtained by suitably immunizing a species of Camelid withHER3 (i.e. so as to raise an immune response and/or heavy chainantibodies directed against HER3), by obtaining a suitable biologicalsample from said Camelid (such as a blood sample, serum sample or sampleof B-cells), and by generating V_(HH) sequences directed against HER3,starting from said sample, using any suitable technique known per se.Such techniques will be clear to the skilled person and/or are furtherdescribed herein.

Alternatively, such naturally occurring V_(HH) domains against HER3, canbe obtained from naïve libraries of Camelid V_(HH) sequences, forexample by screening such a library using HER3, or at least one part,fragment, antigenic determinant or epitope thereof using one or morescreening techniques known per se. Such libraries and techniques are forexample described in WO 99/37681, WO 01/90190, WO 03/025020 and WO03/035694. Alternatively, improved synthetic or semi-synthetic librariesderived from naïve V_(HH) libraries may be used, such as V_(HH)libraries obtained from naïve V_(HH) libraries by techniques such asrandom mutagenesis and/or CDR shuffling, as for example described in WO00/43507.

Thus, in another aspect, the invention relates to a method forgenerating Nanobodies, that are directed against HER3. In one aspect,said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and-   b) screening said set, collection or library of Nanobody sequences    for Nanobody sequences that can bind to and/or have affinity for    HER3;    and-   c) isolating the Nanobody or Nanobodies that can bind to and/or have    affinity for HER3.

In such a method, the set, collection or library of Nanobody sequencesmay be a naïve set, collection or library of Nanobody sequences; asynthetic or semi-synthetic set, collection or library of Nanobodysequences; and/or a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofNanobody sequences may be an immune set, collection or library ofNanobody sequences, and in particular an immune set, collection orlibrary of V_(HH) sequences, that have been derived from a species ofCamelid that has been suitably immunized with HER3 or with a suitableantigenic determinant based thereon or derived therefrom, such as anantigenic part, fragment, region, domain, loop or other epitope thereof.In one particular aspect, said antigenic determinant may be anextracellular part, region, domain, loop or other extracellularepitope(s).

In the above methods, the set, collection or library of Nanobody orV_(HH) sequences may be displayed on a phage, phagemid, ribosome orsuitable micro-organism (such as yeast), such as to facilitatescreening. Suitable methods, techniques and host organisms fordisplaying and screening (a set, collection or library of) Nanobodysequences will be clear to the person skilled in the art, for example onthe basis of the further disclosure herein. Reference is also made tothe review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116(2005).

In another aspect, the method for generating Nanobody sequencescomprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species    of Camelid that express immunoglobulin sequences;-   b) screening said collection or sample of cells for (i) cells that    express an immunoglobulin sequence that can bind to and/or have    affinity for HER3; and (ii) cells that express heavy chain    antibodies, in which substeps (i) and (ii) can be performed    essentially as a single screening step or in any suitable order as    two separate screening steps, so as to provide at least one cell    that expresses a heavy chain antibody that can bind to and/or has    affinity for HER3;    and-   c) either (i) isolating from said cell the V_(HH) sequence present    in said heavy chain antibody; or (ii) isolating from said cell a    nucleic acid sequence that encodes the V_(HH) sequence present in    said heavy chain antibody, followed by expressing said V_(HH)    domain.

In the method according to this aspect, the collection or sample ofcells may for example be a collection or sample of B-cells. Also, inthis method, the sample of cells may be derived from a Camelid that hasbeen suitably immunized with HER3 or a suitable antigenic determinantbased thereon or derived therefrom, such as an antigenic part, fragment,region, domain, loop or other epitope thereof. In one particular aspect,said antigenic determinant may be an extracellular part, region, domain,loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will beclear to the skilled person. Reference is for example made to EP 0 542810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of stepb) is preferably performed using a flow cytometry technique such asFACS. For this, reference is for example made to Lieby et al., Blood,Vol. 97, No. 12, 3820. Particular reference is made to the so-called“Nanoclone®” technique described in International application WO06/079372 by Ablynx N.V.

In another aspect, the method for generating an amino acid sequencedirected against HER3 may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences    encoding heavy chain antibodies or Nanobody sequences;-   b) screening said set, collection or library of nucleic acid    sequences for nucleic acid sequences that encode a heavy chain    antibody or a Nanobody sequence that can bind to and/or has affinity    for HER3;    and-   c) isolating said nucleic acid sequence, followed by expressing the    V_(HH) sequence present in said heavy chain antibody or by    expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acidsequences encoding heavy chain antibodies or Nanobody sequences may forexample be a set, collection or library of nucleic acid sequencesencoding a naïve set, collection or library of heavy chain antibodies orV_(HH) sequences; a set, collection or library of nucleic acid sequencesencoding a synthetic or semi-synthetic set, collection or library ofNanobody sequences; and/or a set, collection or library of nucleic acidsequences encoding a set, collection or library of Nanobody sequencesthat have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library ofnucleic acid sequences may be an immune set, collection or library ofnucleic acid sequences encoding heavy chain antibodies or V_(HH)sequences derived from a Camelid that has been suitably immunized withHER3 or with a suitable antigenic determinant based thereon or derivedtherefrom, such as an antigenic part, fragment, region, domain, loop orother epitope thereof. In one particular aspect, said antigenicdeterminant may be an extracellular part, region, domain, loop or otherextracellular epitope(s).

In the above methods, the set, collection or library of nucleotidesequences may be displayed on a phage, phagemid, ribosome or suitablemicro-organism (such as yeast), such as to facilitate screening.Suitable methods, techniques and host organisms for displaying andscreening (a set, collection or library of) nucleotide sequencesencoding amino acid sequences will be clear to the person skilled in theart, for example on the basis of the further disclosure herein.Reference is also made to WO03054016 and to the review by Hoogenboom inNature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of themethods described herein can also be performed as a selection step.Accordingly the term “screening” as used in the present description cancomprise selection, screening or any suitable combination of selectionand/or screening techniques. Also, when a set, collection or library ofsequences is used, it may contain any suitable number of sequences, suchas 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷,10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collectionor library of amino acid sequences may be obtained or defined byrational, or semi-empirical approaches such as computer modellingtechniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two ormore sequences that are variants from one another (e.g. with designedpoint mutations or with randomized positions), compromise multiplesequences derived from a diverse set of naturally diversified sequences(e.g. an immune library), or any other source of diverse sequences (asdescribed for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection orlibrary of sequences can be displayed on the surface of a phageparticle, a ribosome, a bacterium, a yeast cell, a mammalian cell, andlinked to the nucleotide sequence encoding the amino acid sequencewithin these carriers. This makes such set, collection or libraryamenable to selection procedures to isolate the desired amino acidsequences of the invention. More generally, when a sequence is displayedon a suitable host or host cell, it is also possible (and customary) tofirst isolate from said host or host cell a nucleotide sequence thatencodes the desired sequence, and then to obtain the desired sequence bysuitably expressing said nucleotide sequence in a suitable hostorganism. Again, this can be performed in any suitable manner known perse, as will be clear to the skilled person.

The invention also relates to the V_(HH) sequences or Nanobody sequencesthat are obtained by the above methods, or alternatively by a methodthat comprises the one of the above methods and in addition at least thesteps of determining the nucleotide sequence or amino acid sequence ofsaid V_(HH) sequence or Nanobody sequence; and of expressing orsynthesizing said V_(HH) sequence or Nanobody sequence in a manner knownper se, such as by expression in a suitable host cell or host organismor by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of theinvention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(HH)domain, but that has been e.g. “humanized” or otherwise sequenceoptimized in view of better production yields or better stability, i.e.by replacing one or more amino acid residues in the amino acid sequenceof said naturally occurring V_(HH) sequence (and in particular in theframework sequences) by one or more of the amino acid residues thatoccur at the corresponding position(s) in a V_(H) domain from aconventional 4-chain antibody from a human being (e.g. indicated above),as further described on, and using the techniques mentioned on, page 63of WO 08/020,079. Another particularly preferred class of Nanobodies ofthe invention comprises Nanobodies with an amino acid sequence thatcorresponds to the amino acid sequence of a naturally occurring V_(H)domain, but that has been “camelized”, i.e. by replacing one or moreamino acid residues in the amino acid sequence of a naturally occurringV_(H) domain from a conventional 4-chain antibody by one or more of theamino acid residues that occur at the corresponding position(s) in aV_(HH) domain of a heavy chain antibody, as further described on, andusing the techniques mentioned on, page 63 of WO 08/020,079.

Other suitable methods and techniques for obtaining the Nanobodies ofthe invention and/or nucleic acids encoding the same, starting fromnaturally occurring V_(H) sequences or preferably V_(HH) sequences, willbe clear from the skilled person, and may for example include thetechniques that are mentioned on page 64 of WO 08/00279As mentionedherein, Nanobodies may in particular be characterized by the presence ofone or more “Hallmark residues” (as described herein) in one or more ofthe framework sequences.

The invention in its broadest sense also comprises derivatives of theNanobodies of the invention. Such derivatives can generally be obtainedby modification, and in particular by chemical and/or biological (e.genzymatical) modification, of the Nanobodies of the invention and/or ofone or more of the amino acid residues that form the Nanobodies of theinvention.

Examples of such modifications, as well as examples of amino acidresidues within the Nanobody sequence that can be modified in such amanner (i.e. either on the protein backbone but preferably on a sidechain), methods and techniques that can be used to introduce suchmodifications and the potential uses and advantages of suchmodifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. bycovalent linking or in an other suitable manner) of one or morefunctional groups, residues or moieties into or onto the Nanobody of theinvention, and in particular of one or more functional groups, residuesor moieties that confer one or more desired properties orfunctionalities to the Nanobody of the invention. Example of suchfunctional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. bycovalent binding or in any other suitable manner) of one or morefunctional groups that increase the half-life, the solubility and/or theabsorption of the Nanobody of the invention, that reduce theimmunogenicity and/or the toxicity of the Nanobody of the invention,that eliminate or attenuate any undesirable side effects of the Nanobodyof the invention, and/or that confer other advantageous properties toand/or reduce the undesired properties of the Nanobodies and/orpolypeptides of the invention; or any combination of two or more of theforegoing. Examples of such functional groups and of techniques forintroducing them will be clear to the skilled person, and can generallycomprise all functional groups and techniques mentioned in the generalbackground art cited hereinabove as well as the functional groups andtechniques known per se for the modification of pharmaceutical proteins,and in particular for the modification of antibodies or antibodyfragments (including ScFv's and single domain antibodies), for whichreference is for example made to Remington's Pharmaceutical Sciences,16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functionalgroups may for example be linked directly (for example covalently) to aNanobody of the invention, or optionally via a suitable linker orspacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-lifeand/or reducing the immunogenicity of pharmaceutical proteins comprisesattachment of a suitable pharmacologically acceptable polymer, such aspoly(ethyleneglycol) (PEG) or derivatives thereof (such asmethoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form ofpegylation can be used, such as the pegylation used in the art forantibodies and antibody fragments (including but not limited to (single)domain antibodies and ScFv's); reference is made to for example Chapman,Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. DrugDeliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug.Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylationof proteins are also commercially available, for example from NektarTherapeutics, USA.

Preferably, site-directed pegylation is used, in particular via acysteine-residue (see for example Yang et al., Protein Engineering, 16,10, 761-770 (2003). For example, for this purpose, PEG may be attachedto a cysteine residue that naturally occurs in a Nanobody of theinvention, a Nanobody of the invention may be modified so as to suitablyintroduce one or more cysteine residues for attachment of PEG, or anamino acid sequence comprising one or more cysteine residues forattachment of PEG may be fused to the N- and/or C-terminus of a Nanobodyof the invention, all using techniques of protein engineering known perse to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG isused with a molecular weight of more than 5000, such as more than 10,000and less than 200,000, such as less than 100,000; for example in therange of 20,000-80,000.

Another, usually less preferred modification comprises N-linked orO-linked glycosylation, usually as part of co-translational and/orpost-translational modification, depending on the host cell used forexpressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or moredetectable labels or other signal-generating groups or moieties,depending on the intended use of the labelled Nanobody. Suitable labelsand techniques for attaching, using and detecting them will be clear tothe skilled person, and for example include, but are not limited to, thefluorescent labels, phosphorescent labels, chemiluminescent labels,bioluminescent labels, radio-isotopes, metals, metal chelates, metalliccations, chromophores and enzymes, such as those mentioned on page 109of WO 08/020,079. Other suitable labels will be clear to the skilledperson, and for example include moieties that can be detected using NMRor ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may forexample be used for in vitro, in vivo or in situ assays (includingimmunoassays known per se such as ELISA, RIA, EIA and other “sandwichassays”, etc.) as well as in vivo diagnostic and imaging purposes,depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involvethe introduction of a chelating group, for example to chelate one of themetals or metallic cations referred to above. Suitable chelating groupsfor example include, without limitation, diethyl-enetriaminepentaaceticacid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functionalgroup that is one part of a specific binding pair, such as thebiotin-(strept)avidin binding pair. Such a functional group may be usedto link the Nanobody of the invention to another protein, polypeptide orchemical compound that is bound to the other half of the binding pair,i.e. through formation of the binding pair. For example, a Nanobody ofthe invention may be conjugated to biotin, and linked to anotherprotein, polypeptide, compound or carrier conjugated to avidin orstreptavidin. For example, such a conjugated Nanobody may be used as areporter, for example in a diagnostic system where a detectablesignal-producing agent is conjugated to avidin or streptavidin. Suchbinding pairs may for example also be used to bind the Nanobody of theinvention to a carrier, including carriers suitable for pharmaceuticalpurposes. One non-limiting example are the liposomal formulationsdescribed by Cao and Suresh, Journal of Drug Targetting, 8, 4, 257(2000). Such binding pairs may also be used to link a therapeuticallyactive agent to the Nanobody of the invention.

For some applications, in particular for those applications in which itis intended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation such acell, the Nanobodies of the invention may also be linked to a toxin orto a toxic residue or moiety. Examples of toxic moieties, compounds orresidues which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic compound will be clear to the skilledperson and can for example be found in the prior art cited above and/orin the further description herein. One example is the so-called ADEPT™technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear tothe skilled person. Such modifications may also be introduced forresearch purposes (e.g. to study function-activity relationships).Reference is for example made to Lundblad and Bradshaw, Biotechnol.Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to HER3 with anaffinity (suitably measured and/or expressed as a K_(D)-value (actual orapparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or ak_(off)-rate, or alternatively as an IC₅₀ value, as further describedherein) that is as defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins orpolypeptides that essentially consist of or comprise at least oneNanobody of the invention. By “essentially consist of” is meant that theamino acid sequence of the polypeptide of the invention either isexactly the same as the amino acid sequence of a Nanobody of theinvention or corresponds to the amino acid sequence of a Nanobody of theinvention which has a limited number of amino acid residues, such as1-20 amino acid residues, for example 1-10 amino acid residues andpreferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 aminoacid residues, added at the amino terminal end, at the carboxy terminalend, or at both the amino terminal end and the carboxy terminal end ofthe amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwiseinfluence the (biological) properties of the Nanobody and may or may notadd further functionality to the Nanobody. For example, such amino acidresidues:

-   -   can comprise an N-terminal Met residue, for example as result of        expression in a heterologous host cell or host organism.    -   may form a signal sequence or leader sequence that directs        secretion of the Nanobody from a host cell upon synthesis.        Suitable secretory leader peptides will be clear to the skilled        person, and may be as further described herein. Usually, such a        leader sequence will be linked to the N-terminus of the        Nanobody, although the invention in its broadest sense is not        limited thereto;    -   may form a sequence or signal that allows the Nanobody to be        directed towards and/or to penetrate or enter into specific        organs, tissues, cells, or parts or compartments of cells,        and/or that allows the Nanobody to penetrate or cross a        biological barrier such as a cell membrane, a cell layer such as        a layer of epithelial cells, a tumor including solid tumors, or        the blood-brain-barrier. Examples of such amino acid sequences        will be clear to the skilled person and include those mentioned        in paragraph c) on page 112 of WO 08/020,079.    -   may form a “tag”, for example an amino acid sequence or residue        that allows or facilitates the purification of the Nanobody, for        example using affinity techniques directed against said sequence        or residue. Thereafter, said sequence or residue may be removed        (e.g. by chemical or enzymatical cleavage) to provide the        Nanobody sequence (for this purpose, the tag may optionally be        linked to the Nanobody sequence via a cleavable linker sequence        or contain a cleavable motif). Some preferred, but non-limiting        examples of such residues are multiple histidine residues,        glutatione residues and a myc-tag (see for example SEQ ID NO:31        of WO 06/12282).    -   may be one or more amino acid residues that have been        functionalized and/or that can serve as a site for attachment of        functional groups. Suitable amino acid residues and functional        groups will be clear to the skilled person and include, but are        not limited to, the amino acid residues and functional groups        mentioned herein for the derivatives of the Nanobodies of the        invention.

According to another aspect, a polypeptide of the invention comprises aNanobody of the invention, which is fused at its amino terminal end, atits carboxy terminal end, or both at its amino terminal end and at itscarboxy terminal end to at least one further amino acid sequence, i.e.so as to provide a fusion protein comprising said Nanobody of theinvention and the one or more further amino acid sequences. Such afusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/ordesired amino acid sequences. The further amino acid sequences may ormay not change, alter or otherwise influence the (biological) propertiesof the Nanobody, and may or may not add further functionality to theNanobody or the polypeptide of the invention. Preferably, the furtheramino acid sequence is such that it confers one or more desiredproperties or functionalities to the Nanobody or the polypeptide of theinvention.

For example, the further amino acid sequence may also provide a secondbinding site, which binding site may be directed against any desiredprotein, polypeptide, antigen, antigenic determinant or epitope(including but not limited to the same protein, polypeptide, antigen,antigenic determinant or epitope against which the Nanobody of theinvention is directed, or a different protein, polypeptide, antigen,antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilledperson, and may generally comprise all amino acid sequences that areused in peptide fusions based on conventional antibodies and fragmentsthereof (including but not limited to ScFv's and single domainantibodies). Reference is for example made to the review by Holliger andHudson, Nature Biotechnology, 23, 9, 1126-1136 (2005).

For example, such an amino acid sequence may be an amino acid sequencethat increases the half-life, the solubility, or the absorption, reducesthe immunogenicity or the toxicity, eliminates or attenuates undesirableside effects, and/or confers other advantageous properties to and/orreduces the undesired properties of the polypeptides of the invention,compared to the Nanobody of the invention per se. Some non-limitingexamples of such amino acid sequences are serum proteins, such as humanserum albumin (see for example WO 00/27435) or haptenic molecules (forexample haptens that are recognized by circulating antibodies, see forexample WO 98/22141).

In particular, it has been described in the art that linking fragmentsof immunoglobulins (such as V_(H) domains) to serum albumin or tofragments thereof can be used to increase the half-life. Reference isfor made to WO 00/27435 and WO 01/077137. According to the invention,the Nanobody of the invention is preferably either directly linked toserum albumin (or to a suitable fragment thereof) or via a suitablelinker, and in particular via a suitable peptide linked so that thepolypeptide of the invention can be expressed as a genetic fusion(protein). According to one specific aspect, the Nanobody of theinvention may be linked to a fragment of serum albumin that at leastcomprises the domain III of serum albumin or part thereof. Reference isfor example made to WO 07/112,940 of Ablynx N.V.

Alternatively, the further amino acid sequence may provide a secondbinding site or binding unit that is directed against a serum protein(such as, for example, human serum albumin or another serum protein suchas IgG), so as to provide increased half-life in serum. Such amino acidsequences for example include the Nanobodies described below, as well asthe small peptides and binding proteins described in WO 91/01743, WO01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41);4926-42, 2005, as well as to EP 0 368 684, as well as to WO 08/028,977,WO 08/043,821, WO 08/043,822, WO 2008/068280 and WO 2009/127691).

Such amino acid sequences may in particular be directed against serumalbumin (and more in particular human serum albumin) and/or against IgG(and more in particular human IgG). For example, such amino acidsequences may be amino acid sequences that are directed against (human)serum albumin and amino acid sequences that can bind to amino acidresidues on (human) serum albumin that are not involved in binding ofserum albumin to FcRn (see for example WO 06/0122787) and/or amino acidsequences that are capable of binding to amino acid residues on serumalbumin that do not form part of domain III of serum albumin (see againfor example WO 06/0122787); amino acid sequences that have or canprovide an increased half-life (see for example WO 08/028,977 by AblynxN.V.); amino acid sequences against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus), reference is again made to WO08/028,977; amino acid sequences that can bind to serum albumin in a pHindependent manner (see for example WO 08/043,821 by Ablynx N.V.entitled “Amino acid sequences that bind to serum proteins in a mannerthat is essentially independent of the pH, compounds comprising thesame, and uses thereof”) and/or amino acid sequences that areconditional binders (see for example WO 08/043,822 by Ablynx N.V.entitled “Amino acid sequences that bind to a desired molecule in aconditional manner”).

According to another aspect, the one or more further amino acidsequences may comprise one or more parts, fragments or domains ofconventional 4-chain antibodies (and in particular human antibodies)and/or of heavy chain antibodies. For example, although usually lesspreferred, a Nanobody of the invention may be linked to a conventional(preferably human) V_(H) or V_(L) domain or to a natural or syntheticanalog of a V_(H) or V_(L) domain, again optionally via a linkersequence (including but not limited to other (single) domain antibodies,such as the dAb's described by Ward et al. supra).

The at least one Nanobody may also be linked to one or more (preferablyhuman) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linkersequence. For instance, a Nanobody linked to a suitable C_(H)1 domaincould for example be used—together with suitable light chains—togenerate antibody fragments/structures analogous to conventional Fabfragments or F(ab′)₂ fragments, but in which one or (in case of anF(ab′)₂ fragment) one or both of the conventional V_(H) domains havebeen replaced by a Nanobody of the invention. Also, two Nanobodies couldbe linked to a C_(H)3 domain (optionally via a linker) to provide aconstruct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, oneor more Nanobodies of the invention may be linked (optionally via asuitable linker or hinge region) to one or more constant domains (forexample, 2 or 3 constant domains that can be used as part of/to form anFc portion), to an Fc portion and/or to one or more antibody parts,fragments or domains that confer one or more effector functions to thepolypeptide of the invention and/or may confer the ability to bind toone or more Fc receptors. For example, for this purpose, and withoutbeing limited thereto, the one or more further amino acid sequences maycomprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, suchas from a heavy chain antibody (as described herein) and more preferablyfrom a conventional human 4-chain antibody; and/or may form (part of)and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 orIgG4), from IgE or from another human Ig such as IgA, IgD or IgM. Forexample, WO 94/04678 describes heavy chain antibodies comprising aCamelid V_(HH) domain or a humanized derivative thereof (i.e. aNanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domain have beenreplaced by human C_(H)2 and C_(H)3 domains, so as to provide animmunoglobulin that consists of 2 heavy chains each comprising aNanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain),which immunoglobulin has the effector function provided by the C_(H)2and C_(H)3 domains and which immunoglobulin can function without thepresence of any light chains. Other amino acid sequences that can besuitably linked to the Nanobodies of the invention so as to provide aneffector function will be clear to the skilled person, and may be chosenon the basis of the desired effector function(s). Reference is forexample made to WO 04/058820, WO 99/42077, WO 02/056910 and WO05/017148, as well as the review by Holliger and Hudson, supra; and tothe non-prepublished US provisional application by Ablynx N.V. entitled“Constructs comprising single variable domains and an Fc portion derivedfrom IgE” which has a filing date of Dec. 4, 2007. Coupling of aNanobody of the invention to an Fc portion may also lead to an increasedhalf-life, compared to the corresponding Nanobody of the invention. Forsome applications, the use of an Fc portion and/or of constant domains(i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-lifewithout any biologically significant effector function may also besuitable or even preferred. Other suitable constructs comprising one ormore Nanobodies and one or more constant domains with increasedhalf-life in vivo will be clear to the skilled person, and may forexample comprise two Nanobodies linked to a C_(H)3 domain, optionallyvia a linker sequence. Generally, any fusion protein or derivatives withincreased half-life will preferably have a molecular weight of more than50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form apolypeptide of the invention, one or more amino acid sequences of theinvention may be linked (optionally via a suitable linker or hingeregion) to naturally occurring, synthetic or semisynthetic constantdomains (or analogs, variants, mutants, parts or fragments thereof) thathave a reduced (or essentially no) tendency to self-associate intodimers (i.e. compared to constant domains that naturally occur inconventional 4-chain antibodies). Such monomeric (i.e. notself-associating) Fc chain variants, or fragments thereof, will be clearto the skilled person. For example, Helm et al., J Biol Chem 1996 2717494, describe monomeric Fcϵ chain variants that can be used in thepolypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they arestill capable of binding to the complement or the relevant Fcreceptor(s) (depending on the Fc portion from which they are derived),and/or such that they still have some or all of the effector functionsof the Fc portion from which they are derived (or at a reduced levelstill suitable for the intended use). Alternatively, in such apolypeptide chain of the invention, the monomeric Fc chain may be usedto confer increased half-life upon the polypeptide chain, in which casethe monomeric Fc chain may also have no or essentially no effectorfunctions.

Bivalent/multivalent, bispecific/multispecific orbiparatopic/multiparatopic polypeptides of the invention may also belinked to Fc portions, in order to provide polypeptide constructs of thetype that is described in the non-prepublished U.S. provisionalapplication U.S. 61/005,331 entitled “immunoglobulin constructs” filedon Dec. 4, 2007.

The further amino acid sequences may also form a signal sequence orleader sequence that directs secretion of the Nanobody or thepolypeptide of the invention from a host cell upon synthesis (forexample to provide a pre-, pro- or prepro-form of the polypeptide of theinvention, depending on the host cell used to express the polypeptide ofthe invention).

The further amino acid sequence may also form a sequence or signal thatallows the Nanobody or polypeptide of the invention to be directedtowards and/or to penetrate or enter into specific organs, tissues,cells, or parts or compartments of cells, and/or that allows theNanobody or polypeptide of the invention to penetrate or cross abiological barrier such as a cell membrane, a cell layer such as a layerof epithelial cells, a tumor including solid tumors, or theblood-brain-barrier. Suitable examples of such amino acid sequences willbe clear to the skilled person, and for example include, but are notlimited to, those mentioned on page 118 of WO 08/020,079. For someapplications, in particular for those applications in which it isintended to kill a cell that expresses the target against which theNanobodies of the invention are directed (e.g. in the treatment ofcancer), or to reduce or slow the growth and/or proliferation of such acell, the Nanobodies of the invention may also be linked to a(cyto)toxic protein or polypeptide. Examples of such toxic proteins andpolypeptides which can be linked to a Nanobody of the invention toprovide—for example—a cytotoxic polypeptide of the invention will beclear to the skilled person and can for example be found in the priorart cited above and/or in the further description herein. One example isthe so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or morefurther amino acid sequences comprise at least one further Nanobody, soas to provide a polypeptide of the invention that comprises at leasttwo, such as three, four, five or more Nanobodies, in which saidNanobodies may optionally be linked via one or more linker sequences (asdefined herein). As described on pages 119 and 120 of WO 08/020,079,polypeptides of the invention that comprise two or more Nanobodies, ofwhich at least one is a Nanobody of the invention, will also be referredto herein as “multivalent” polypeptides of the invention, and theNanobodies present in such polypeptides will also be referred to hereinas being in a “multivalent format”. For example, “bivalent” and“trivalent” polypeptides of the invention may be as further described onpages 119 and 120 of WO 08/020,079.

Polypeptides of the invention that contain at least two Nanobodies, inwhich at least one Nanobody is directed against a first antigen (i.e.against HER3) and at least one Nanobody is directed against a secondantigen (i.e. different from HER3), will also be referred to as“multispecific” polypeptides of the invention, and the Nanobodiespresent in such polypeptides will also be referred to herein as being ina “multispecific format”. Thus, for example, a “bispecific” polypeptideof the invention is a polypeptide that comprises at least one Nanobodydirected against a first antigen (i.e. HER3) and at least one furtherNanobody directed against a second antigen (i.e. different from HER3),whereas a “trispecific” polypeptide of the invention is a polypeptidethat comprises at least one Nanobody directed against a first antigen(i.e. HER3), at least one further Nanobody directed against a secondantigen (i.e. different from HER3) and at least one further Nanobodydirected against a third antigen (i.e. different from both HER3, and thesecond antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of theinvention is a bivalent polypeptide of the invention (as definedherein), comprising a first Nanobody directed against HER3, and a secondNanobody directed against a second antigen, in which said first andsecond Nanobody may optionally be linked via a linker sequence (asdefined herein); whereas a trispecific polypeptide of the invention inits simplest form is a trivalent polypeptide of the invention (asdefined herein), comprising a first Nanobody directed against HER3, asecond Nanobody directed against a second antigen and a third Nanobodydirected against a third antigen, in which said first, second and thirdNanobody may optionally be linked via one or more, and in particular oneand more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, theinvention is not limited thereto, in the sense that a multispecificpolypeptide of the invention may comprise at least one Nanobody againstHER3, and any number of Nanobodies directed against one or more antigensdifferent from HER3.

Furthermore, although it is encompassed within the scope of theinvention that the specific order or arrangement of the variousNanobodies in the polypeptides of the invention may have some influenceon the properties of the final polypeptide of the invention (includingbut not limited to the affinity, specificity or avidity for HER3, oragainst the one or more other antigens), said order or arrangement isusually not critical and may be suitably chosen by the skilled person,optionally after some limited routine experiments based on thedisclosure herein. Thus, when reference is made to a specificmultivalent or multispecific polypeptide of the invention, it should benoted that this encompasses any order or arrangements of the relevantNanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that thepolypeptides of the invention contain two or more Nanobodies and one ormore further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or moreV_(HH) domains and their preparation, reference is also made to Conrathet al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans,Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to forexample WO 96/34103 and WO 99/23221. Some other examples of somespecific multispecific and/or multivalent polypeptide of the inventioncan be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptideof the invention comprises at least one Nanobody of the invention and atleast one Nanobody that provides for an increased half-life. SuchNanobodies may for example be Nanobodies that are directed against aserum protein, and in particular a human serum protein, such as humanserum albumin, thyroxine-binding protein, (human) transferrin,fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one ofthe serum proteins listed in WO 04/003019. Of these, Nanobodies that canbind to serum albumin (and in particular human serum albumin) or to IgG(and in particular human IgG, see for example Nanobody VH-1 described inthe review by Muyldermans, supra) are particularly preferred (althoughfor example, for experiments in mice or primates, Nanobodies against orcross-reactive with mouse serum albumin (MSA) or serum albumin from saidprimate, respectively, can be used. However, for pharmaceutical use,Nanobodies against human serum albumin or human IgG will usually bepreferred). Nanobodies that provide for increased half-life and that canbe used in the polypeptides of the invention include the Nanobodiesdirected against serum albumin that are described in WO 04/041865, in WO06/122787 and in the further patent applications by Ablynx N.V., such asthose mentioned above.

For example, some preferred Nanobodies that provide for increasedhalf-life for use in the present invention include Nanobodies that canbind to amino acid residues on (human) serum albumin that are notinvolved in binding of serum albumin to FcRn (see for example WO06/0122787); Nanobodies that are capable of binding to amino acidresidues on serum albumin that do not form part of domain III of serumalbumin (see for example WO 06/0122787); Nanobodies that have or canprovide an increased half-life (see for example WO 08/028,977 by AblynxN.V mentioned herein); Nanobodies against human serum albumin that arecross-reactive with serum albumin from at least one species of mammal,and in particular with at least one species of primate (such as, withoutlimitation, monkeys from the genus Macaca (such as, and in particular,cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macacamulatta)) and baboon (Papio ursinus)) (see for example WO 08/028,977 byAblynx N.V)); Nanobodies that can bind to serum albumin in a pHindependent manner (see for example WO2008/043821 by Ablynx N.V.mentioned herein) and/or Nanobodies that are conditional binders (seefor example WO 08/043,822 by Ablynx N.V.).

Some particularly preferred Nanobodies that provide for increasedhalf-life and that can be used in the polypeptides of the inventioninclude the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (seeTables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787, seealso SEQ ID NO: 11 of this application) is particularly preferred.

Some preferred, but non-limiting examples of polypeptides of theinvention that comprise at least one Nanobody of the invention and atleast one Nanobody that provides for increased half-life are given inSEQ ID NO's 147 to 327, more preferably HER3MS00135 (SEQ ID NO:282),HER3MS00212 (SEQ ID NO:319) or HER3MS00215 (SEQ ID NO:322).

According to a specific, but non-limiting aspect of the invention, thepolypeptides of the invention contain, besides the one or moreNanobodies of the invention, at least one Nanobody against human serumalbumin.

Generally, any polypeptides of the invention with increased half-lifethat contain one or more Nanobodies of the invention, and anyderivatives of Nanobodies of the invention or of such polypeptides thathave an increased half-life, preferably have a half-life that is atleast 1.5 times, preferably at least 2 times, such as at least 5 times,for example at least 10 times or more than 20 times, greater than thehalf-life of the corresponding Nanobody of the invention per se. Forexample, such a derivative or polypeptides with increased half-life mayhave a half-life that is increased with more than 1 hours, preferablymore than 2 hours, more preferably more than 6 hours, such as more than12 hours, or even more than 24, 48 or 72 hours, compared to thecorresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, suchderivatives or polypeptides may exhibit a serum half-life in human of atleast about 12 hours, preferably at least 24 hours, more preferably atleast 48 hours, even more preferably at least 72 hours or more. Forexample, such derivatives or polypeptides may have a half-life of atleast 5 days (such as about 5 to 10 days), preferably at least 9 days(such as about 9 to 14 days), more preferably at least about 10 days(such as about 10 to 15 days), or at least about 11 days (such as about11 to 16 days), more preferably at least about 12 days (such as about 12to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable ofbinding to one or more molecules which can increase the half-life of thepolypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo.

In the polypeptides of the invention, the one or more Nanobodies and theone or more polypeptides may be directly linked to each other (as forexample described in WO 99/23221) and/or may be linked to each other viaone or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecificpolypeptides will be clear to the skilled person, and may generally beany linker or spacer used in the art to link amino acid sequences.Preferably, said linker or spacer is suitable for use in constructingproteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers thatare used in the art to link antibody fragments or antibody domains.These include the linkers mentioned in the general background art citedabove, as well as for example linkers that are used in the art toconstruct diabodies or ScFv fragments (in this respect, however, itsshould be noted that, whereas in diabodies and in ScFv fragments, thelinker sequence used should have a length, a degree of flexibility andother properties that allow the pertinent V_(H) and V_(L) domains tocome together to form the complete antigen-binding site, there is noparticular limitation on the length or the flexibility of the linkerused in the polypeptide of the invention, since each Nanobody by itselfforms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and inparticular amino acid sequences of between 1 and 50, preferably between1 and 30, such as between 1 and 10 amino acid residues. Some preferredexamples of such amino acid sequences include gly-ser linkers, forexample of the type (gly_(x)ser_(y))_(z), such as (for example(gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30,GS15, GS9 and GS7 linkers described in the applications by Ablynxmentioned herein (see for example WO 06/040153 and WO 06/122825), aswell as hinge-like regions, such as the hinge regions of naturallyoccurring heavy chain antibodies or similar sequences (such as describedin WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such asAAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) andGS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers,in particular those suitable for use in proteins for pharmaceutical use.For instance, poly(ethyleneglycol) moieties have been used to linkantibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, thedegree of flexibility and/or other properties of the linker(s) used(although not critical, as it usually is for linkers used in ScFvfragments) may have some influence on the properties of the finalpolypeptide of the invention, including but not limited to the affinity,specificity or avidity for HER3, or for one or more of the otherantigens. Based on the disclosure herein, the skilled person will beable to determine the optimal linker(s) for use in a specificpolypeptide of the invention, optionally after some limited routineexperiments.

For example, in multivalent polypeptides of the invention that compriseNanobodies directed against a multimeric antigen (such as a multimericreceptor or other protein), the length and flexibility of the linker arepreferably such that it allows each Nanobody of the invention present inthe polypeptide to bind to the antigenic determinant on each of thesubunits of the multimer. Similarly, in a multispecific polypeptide ofthe invention that comprises Nanobodies directed against two or moredifferent antigenic determinants on the same antigen (for exampleagainst different epitopes of an antigen and/or against differentsubunits of a multimeric receptor, channel or protein), the length andflexibility of the linker are preferably such that it allows eachNanobody to bind to its intended antigenic determinant. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linker(s) for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) usedconfer one or more other favourable properties or functionality to thepolypeptides of the invention, and/or provide one or more sites for theformation of derivatives and/or for the attachment of functional groups(e.g. as described herein for the derivatives of the Nanobodies of theinvention). For example, linkers containing one or more charged aminoacid residues (see Table A-2 on page 48 of the International applicationWO 08/020,079) can provide improved hydrophilic properties, whereaslinkers that form or contain small epitopes or tags can be used for thepurposes of detection, identification and/or purification. Again, basedon the disclosure herein, the skilled person will be able to determinethe optimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of theinvention, these linkers may be the same or different. Again, based onthe disclosure herein, the skilled person will be able to determine theoptimal linkers for use in a specific polypeptide of the invention,optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of theinvention will be a linear polypeptide. However, the invention in itsbroadest sense is not limited thererto. For example, when a polypeptideof the invention comprises three of more Nanobodies, it is possible tolink them by use of a linker with three or more “arms”, which each “arm”being linked to a Nanobody, so as to provide a “star-shaped” construct.It is also possible, although usually less preferred, to use circularconstructs.

The invention also comprises derivatives of the polypeptides of theinvention, which may be essentially analogous to the derivatives of theNanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentiallyconsist” of a polypeptide of the invention (in which the wording“essentially consist of” has essentially the same meaning as indicatedabove).

According to one aspect of the invention, the polypeptide of theinvention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids ofthe invention can be prepared in a manner known per se, as will be clearto the skilled person from the further description herein. For example,the Nanobodies and polypeptides of the invention can be prepared in anymanner known per se for the preparation of antibodies and in particularfor the preparation of antibody fragments (including but not limited to(single) domain antibodies and ScFv fragments). Some preferred, butnon-limiting methods for preparing the amino acid sequences, ISV's,Nanobodies, polypeptides and nucleic acids include the methods andtechniques described herein.

As will be clear to the skilled person, one particularly useful methodfor preparing an amino acid sequence, ISV, Nanobody and/or a polypeptideof the invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also    referred to herein as a “host of the invention”) or in another    suitable expression system of a nucleic acid that encodes said amino    acid sequence, ISV, Nanobody or polypeptide of the invention (also    referred to herein as a “nucleic acid of the invention”), optionally    followed by:-   ii) isolating and/or purifying the amino acid sequence, ISV,    Nanobody or polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under    conditions that are such that said host of the invention expresses    and/or produces at least one amino acid sequence, ISV, Nanobody    and/or polypeptide of the invention; optionally followed by:-   ii) isolating and/or purifying the amino acid sequence, ISV,    Nanobody or polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or doublestranded DNA or RNA, and is preferably in the form of double strandedDNA. For example, the nucleotide sequences of the invention may begenomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage thathas been specifically adapted for expression in the intended host cellor host organism).

According to one aspect of the invention, the nucleic acid of theinvention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a vector, such as for example a plasmid, cosmid orYAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in amanner known per se, based on the information on the amino acidsequences for the polypeptides of the invention given herein, and/or canbe isolated from a suitable natural source. To provide analogs,nucleotide sequences encoding naturally occurring V_(HH) domains can forexample be subjected to site-directed mutagenesis, so at to provide anucleic acid of the invention encoding said analog. Also, as will beclear to the skilled person, to prepare a nucleic acid of the invention,also several nucleotide sequences, such as at least one nucleotidesequence encoding a Nanobody and for example nucleic acids encoding oneor more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will beclear to the skilled person and may for instance include, but are notlimited to, automated DNA synthesis; site-directed mutagenesis;combining two or more naturally occurring and/or synthetic sequences (ortwo or more parts thereof), introduction of mutations that lead to theexpression of a truncated expression product; introduction of one ormore restriction sites (e.g. to create cassettes and/or regions that mayeasily be digested and/or ligated using suitable restriction enzymes),and/or the introduction of mutations by means of a PCR reaction usingone or more “mismatched” primers, using for example a sequence of anaturally occurring form of HER3 as a template. These and othertechniques will be clear to the skilled person, and reference is againmade to the standard handbooks, such as Sambrook et al. and Ausubel etal., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be presentin and/or be part of a genetic construct, as will be clear to the personskilled in the art and as described on pages 131-134 of WO 08/020,079(incorporated herein by reference). Such genetic constructs generallycomprise at least one nucleic acid of the invention that is optionallylinked to one or more elements of genetic constructs known per se, suchas for example one or more suitable regulatory elements (such as asuitable promoter(s), enhancer(s), terminator(s), etc.) and the furtherelements of genetic constructs referred to herein. Such geneticconstructs comprising at least one nucleic acid of the invention willalso be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and arepreferably double-stranded DNA. The genetic constructs of the inventionmay also be in a form suitable for transformation of the intended hostcell or host organism, in a form suitable for integration into thegenomic DNA of the intended host cell or in a form suitable forindependent replication, maintenance and/or inheritance in the intendedhost organism. For instance, the genetic constructs of the invention maybe in the form of a vector, such as for example a plasmid, cosmid, YAC,a viral vector or transposon. In particular, the vector may be anexpression vector, i.e. a vector that can provide for expression invitro and/or in vivo (e.g. in a suitable host cell, host organism and/orexpression system).

In a preferred but non-limiting aspect, a genetic construct of theinvention comprises

-   i) at least one nucleic acid of the invention; operably connected to-   ii) one or more regulatory elements, such as a promoter and    optionally a suitable terminator;    and optionally also-   iii) one or more further elements of genetic constructs known per    se;

in which the terms “operably connected” and “operably linked” have themeaning given on pages 131-134 of WO 08/020,079; and in which the“regulatory elements”, “promoter”, “terminator” and “further elements”are as described on pages 131-134 of WO 08/020,079; and in which thegenetic constructs may further be as described on pages 131-134 of WO08/020,079.

The nucleic acids of the invention and/or the genetic constructs of theinvention may be used to transform a host cell or host organism, i.e.for expression and/or production of the amino acid sequence, ISV,Nanobody or polypeptide of the invention. Suitable hosts or host cellswill be clear to the skilled person, and may for example be any suitablefungal, prokaryotic or eukaryotic cell or cell line or any suitablefungal, prokaryotic or eukaryotic organism, for example those describedon pages 134 and 135 of WO 08/020,079; as well as all other hosts orhost cells known per se for the expression and production of antibodiesand antibody fragments (including but not limited to (single) domainantibodies and ScFv fragments), which will be clear to the skilledperson. Reference is also made to the general background art citedhereinabove, as well as to for example WO 94/29457; WO 96/34103; WO99/42077; Frenken et al., (1998), supra; Riechmann and Muyldermans,(1999), supra; van der Linden, (2000), supra; Thomassen et al., (2002),supra; Joosten et al., (2003), supra; Joosten et al., (2005), supra; andthe further references cited herein.

The amino acid sequences, Nanobodies and polypeptides of the inventioncan also be introduced and expressed in one or more cells, tissues ororgans of a multicellular organism, for example for prophylactic and/ortherapeutic purposes (e.g. as a gene therapy), as further described onpages 135 and 136 of in WO 08/020,079, and in the further referencescited in WO 08/020,079.

For expression of the ISV or Nanobodies in a cell, they may also beexpressed as so-called “intrabodies”, as for example described in WO94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; inCattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Developmentand Applications. Landes and Springer-Verlag; and in Kontermann, Methods34, (2004), 163-170.

The amino acid sequences, ISV, Nanobodies and polypeptides of theinvention can for example also be produced in the milk of transgenicmammals, for example in the milk of rabbits, cows, goats or sheep (seefor example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S.Pat. No. 6,849,992 for general techniques for introducing transgenesinto mammals), in plants or parts of plants including but not limited totheir leaves, flowers, fruits, seed, roots or turbers (for example intobacco, maize, soybean or alfalfa) or in for example pupae of thesilkworm Bombix mori.

Furthermore, the amino acid sequences, ISV, Nanobodies and polypeptidesof the invention can also be expressed and/or produced in cell-freeexpression systems, and suitable examples of such systems will be clearto the skilled person. Some preferred, but non-limiting examples includeexpression in the wheat germ system; in rabbit reticulocyte lysates; orin the E. coli Zubay system.

As mentioned above, one of the advantages of the use of ISV orNanobodies is that the polypeptides based thereon can be preparedthrough expression in a suitable bacterial system, and suitablebacterial expression systems, vectors, host cells, regulatory elements,etc., will be clear to the skilled person, for example from thereferences cited above. It should however be noted that the invention inits broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expressionsystem, such as a bacterial expression system, is used that provides thepolypeptides of the invention in a form that is suitable forpharmaceutical use, and such expression systems will again be clear tothe skilled person. As also will be clear to the skilled person,polypeptides of the invention suitable for pharmaceutical use can beprepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the(industrial) production of ISV's, Nanobodies or Nanobody-containingprotein therapeutics include strains of E. coli, Pichia pastoris, S.cerevisiae that are suitable for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical (i.e. GMP grade) expression/production/fermentation.Suitable examples of such strains will be clear to the skilled person.Such strains and production/expression systems are also made availableby companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary(CHO) cells, can be used for large scaleexpression/production/fermentation, and in particular for large scalepharmaceutical expression/production/fermentation. Again, suchexpression/production systems are also made available by some of thecompanies mentioned above.

The choice of the specific expression system would depend in part on therequirement for certain post-translational modifications, morespecifically glycosylation. The production of an ISV- orNanobody-containing recombinant protein for which glycosylation isdesired or required would necessitate the use of mammalian expressionhosts that have the ability to glycosylate the expressed protein. Inthis respect, it will be clear to the skilled person that theglycosylation pattern obtained (i.e. the kind, number and position ofresidues attached) will depend on the cell or cell line that is used forthe expression. Preferably, either a human cell or cell line is used(i.e. leading to a protein that essentially has a human glycosylationpattern) or another mammalian cell line is used that can provide aglycosylation pattern that is essentially and/or functionally the sameas human glycosylation or at least mimics human glycosylation.Generally, prokaryotic hosts such as E. coli do not have the ability toglycosylate proteins, and the use of lower eukaryotes such as yeastusually leads to a glycosylation pattern that differs from humanglycosylation. Nevertheless, it should be understood that all theforegoing host cells and expression systems can be used in theinvention, depending on the desired amino acid sequence, ISV, Nanobodyor polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the aminoacid sequence, ISV, Nanobody or polypeptide of the invention isglycosylated. According to another non-limiting aspect of the invention,the amino acid sequence, ISV, Nanobody or polypeptide of the inventionis non-glycosylated.

According to one preferred, but non-limiting aspect of the invention,the amino acid sequence, ISV, Nanobody or polypeptide of the inventionis produced in a bacterial cell, in particular a bacterial cell suitablefor large scale pharmaceutical production, such as cells of the strainsmentioned above.

According to another preferred, but non-limiting aspect of theinvention, the amino acid sequence, ISV, Nanobody or polypeptide of theinvention is produced in a yeast cell, in particular a yeast cellsuitable for large scale pharmaceutical production, such as cells of thespecies mentioned above.

According to yet another preferred, but non-limiting aspect of theinvention, the amino acid sequence, ISV, Nanobody or polypeptide of theinvention is produced in a mammalian cell, in particular in a human cellor in a cell of a human cell line, and more in particular in a humancell or in a cell of a human cell line that is suitable for large scalepharmaceutical production, such as the cell lines mentioned hereinabove.

As further described on pages 138 and 139 of WO 08/020,079, whenexpression in a host cell is used to produce the amino acid sequences,ISV's, Nanobodies and the polypeptides of the invention, the amino acidsequences, ISV's, Nanobodies and polypeptides of the invention can beproduced either intracellullarly (e.g. in the cytosol, in the periplasmaor in inclusion bodies) and then isolated from the host cells andoptionally further purified; or can be produced extracellularly (e.g. inthe medium in which the host cells are cultured) and then isolated fromthe culture medium and optionally further purified. Thus, according toone non-limiting aspect of the invention, the amino acid sequence, ISV,Nanobody or polypeptide of the invention is an amino acid sequence, ISV,Nanobody or polypeptide that has been produced intracellularly and thathas been isolated from the host cell, and in particular from a bacterialcell or from an inclusion body in a bacterial cell. According to anothernon-limiting aspect of the invention, the amino acid sequence, ISV,Nanobody or polypeptide of the invention is an amino acid sequence, ISV,Nanobody or polypeptide that has been produced extracellularly, and thathas been isolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cellsinclude those mentioned on pages 139 and 140 of WO 08/020,079.

Some preferred, but non-limiting secretory sequences for use with thesehost cells include those mentioned on page 140 of WO 08/020,079.

Suitable techniques for transforming a host or host cell of theinvention will be clear to the skilled person and may depend on theintended host cell/host organism and the genetic construct to be used.Reference is again made to the handbooks and patent applicationsmentioned above.

After transformation, a step for detecting and selecting those hostcells or host organisms that have been successfully transformed with thenucleotide sequence/genetic construct of the invention may be performed.This may for instance be a selection step based on a selectable markerpresent in the genetic construct of the invention or a step involvingthe detection of the amino acid sequence of the invention, e.g. usingspecific antibodies.

The transformed host cell (which may be in the form or a stable cellline) or host organisms (which may be in the form of a stable mutantline or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that theyexpress, or are (at least) capable of expressing (e.g. under suitableconditions), an amino acid sequence, ISV, Nanobody or polypeptide of theinvention (and in case of a host organism: in at least one cell, part,tissue or organ thereof). The invention also includes furthergenerations, progeny and/or offspring of the host cell or host organismof the invention, that may for instance be obtained by cell division orby sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of theinvention, the transformed host cell or transformed host organism maygenerally be kept, maintained and/or cultured under conditions such thatthe (desired) amino acid sequence, ISV, Nanobody or polypeptide of theinvention is expressed/produced. Suitable conditions will be clear tothe skilled person and will usually depend upon the host cell/hostorganism used, as well as on the regulatory elements that control theexpression of the (relevant) nucleotide sequence of the invention.Again, reference is made to the handbooks and patent applicationsmentioned above in the paragraphs on the genetic constructs of theinvention.

Generally, suitable conditions may include the use of a suitable medium,the presence of a suitable source of food and/or suitable nutrients, theuse of a suitable temperature, and optionally the presence of a suitableinducing factor or compound (e.g. when the nucleotide sequences of theinvention are under the control of an inducible promoter); all of whichmay be selected by the skilled person. Again, under such conditions, theamino acid sequences of the invention may be expressed in a constitutivemanner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acidsequence, ISV, Nanobody or polypeptide of the invention may (first) begenerated in an immature form (as mentioned above), which may then besubjected to post-translational modification, depending on the hostcell/host organism used. Also, the amino acid sequence, ISV, Nanobody orpolypeptide of the invention may be glycosylated, again depending on thehost cell/host organism used.

The amino acid sequence, ISV, Nanobody or polypeptide of the inventionmay then be isolated from the host cell/host organism and/or from themedium in which said host cell or host organism was cultivated, usingprotein isolation and/or purification techniques known per se, such as(preparative) chromatography and/or electrophoresis techniques,differential precipitation techniques, affinity techniques (e.g. using aspecific, cleavable amino acid sequence fused with the amino acidsequence, ISV, Nanobody or polypeptide of the invention) and/orpreparative immunological techniques (i.e. using antibodies against theamino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention maybe formulated as a pharmaceutical preparation or compositions comprisingat least one polypeptide of the invention and at least onepharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activepolypeptides and/or compounds. By means of non-limiting examples, such aformulation may be in a form suitable for oral administration, forparenteral administration (such as by intravenous, intramuscular orsubcutaneous injection or intravenous infusion), for topicaladministration, for administration by inhalation, by a skin patch, by animplant, by a suppository, etc. Such suitable administration forms—whichmay be solid, semi-solid or liquid, depending on the manner ofadministration—as well as methods and carriers for use in thepreparation thereof, will be clear to the skilled person, and arefurther described herein.

Thus, in a further aspect, the invention relates to a pharmaceuticalcomposition that contains at least one amino acid of the invention, atleast one ISV of the invention, at least one Nanobody of the inventionor at least one polypeptide of the invention and at least one suitablecarrier, diluent or excipient (i.e. suitable for pharmaceutical use),and optionally one or more further active substances.

Generally, the amino acid sequences, ISV's, Nanobodies and polypeptidesof the invention can be formulated and administered in any suitablemanner known per se, for which reference is for example made to thegeneral background art cited above (and in particular to WO 04/041862,WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/020,079) as well asto the standard handbooks, such as Remington's Pharmaceutical Sciences,18^(th) Ed., Mack Publishing Company, USA (1990), Remington, the Scienceand Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins(2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.),Wiley, Weinheim, 2007 (see for example pages 252-255).

For example, the amino acid sequences, ISV's, Nanobodies andpolypeptides of the invention may be formulated and administered in anymanner known per se for conventional antibodies and antibody fragments(including ScFv's and diabodies) and other pharmaceutically activeproteins. Such formulations and methods for preparing the same will beclear to the skilled person, and for example include preparationssuitable for parenteral administration (for example intravenous,intraperitoneal, subcutaneous, intramuscular, intraluminal,intra-arterial or intrathecal administration) or for topical (i.e.transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterilesolutions, suspensions, dispersions or emulsions that are suitable forinfusion or injection. Suitable carriers or diluents for suchpreparations for example include, without limitation, those mentioned onpage 143 of WO 08/020,079. Usually, aqueous solutions or suspensionswill be preferred.

The amino acid sequences, ISV's, Nanobodies and polypeptides of theinvention can also be administered using gene therapy methods ofdelivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated byreference in its entirety. Using a gene therapy method of delivery,primary cells transfected with the gene encoding an amino acid sequence,ISV, Nanobody or polypeptide of the invention can additionally betransfected with tissue specific promoters to target specific organs,tissue, grafts, tumors, or cells and can additionally be transfectedwith signal and stabilization sequences for subcellularly localizedexpression.

Thus, the amino acid sequences, ISV's, Nanobodies and polypeptides ofthe invention may be systemically administered, e.g., orally, incombination with a pharmaceutically acceptable vehicle such as an inertdiluent or an assimilable edible carrier. They may be enclosed in hardor soft shell gelatin capsules, may be compressed into tablets, or maybe incorporated directly with the food of the patient's diet. For oraltherapeutic administration, the amino acid sequences, ISV's, Nanobodiesand polypeptides of the invention may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% of theamino acid sequence, Nanobody or polypeptide of the invention. Theirpercentage in the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the amino acidsequence, ISV, Nanobody or polypeptide of the invention in suchtherapeutically useful compositions is such that an effective dosagelevel will be obtained.

The tablets, troches, pills, capsules, and the like may also containbinders, excipients, disintegrating agents, lubricants and sweetening orflavouring agents, for example those mentioned on pages 143-144 of WO08/020,079. When the unit dosage form is a capsule, it may contain, inaddition to materials of the above type, a liquid carrier, such as avegetable oil or a polyethylene glycol. Various other materials may bepresent as coatings or to otherwise modify the physical form of thesolid unit dosage form. For instance, tablets, pills, or capsules may becoated with gelatin, wax, shellac or sugar and the like. A syrup orelixir may contain the amino acid sequences, ISV's, Nanobodies andpolypeptides of the invention, sucrose or fructose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any unit dosage form should be pharmaceutically acceptable andsubstantially non-toxic in the amounts employed. In addition, the aminoacid sequences, ISV's, Nanobodies and polypeptides of the invention maybe incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also beprovided with an enteric coating that will allow the constructs of theinvention to resist the gastric environment and pass into theintestines. More generally, preparations and formulations for oraladministration may be suitably formulated for delivery into any desiredpart of the gastrointestinal tract. In addition, suitable suppositoriesmay be used for delivery into the gastrointestinal tract.

The amino acid sequences, ISV's, Nanobodies and polypeptides of theinvention may also be administered intravenously or intraperitoneally byinfusion or injection, as further described on pages 144 and 145 of WO08/020,079.

For topical administration, the amino acid sequences, ISV's, Nanobodiesand polypeptides of the invention may be applied in pure form, i.e.,when they are liquids. However, it will generally be desirable toadminister them to the skin as compositions or formulations, incombination with a dermatologically acceptable carrier, which may be asolid or a liquid, as further described on page 145 of WO 08/020,079.

Generally, the concentration of the amino acid sequences, ISV's,Nanobodies and polypeptides of the invention in a liquid composition,such as a lotion, will be from about 0.1-25 wt-%, preferably from about0.5-10 wt-%. The concentration in a semi-solid or solid composition suchas a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5wt-%.

The amount of the amino acid sequences, ISV's, Nanobodies andpolypeptides of the invention required for use in treatment will varynot only with the particular amino acid sequence, ISV, Nanobody orpolypeptide selected but also with the route of administration, thenature of the condition being treated and the age and condition of thepatient and will be ultimately at the discretion of the attendantphysician or clinician. Also the dosage of the amino acid sequences,ISV's, Nanobodies and polypeptides of the invention varies depending onthe target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By“long-term” is meant at least two weeks and preferably, several weeks,months, or years of duration. Necessary modifications in this dosagerange may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. See Remington'sPharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co.,Easton, Pa. The dosage can also be adjusted by the individual physicianin the event of any complication.

In another aspect, the invention relates to a method for the preventionand/or treatment of at least one variety of cancers, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of an ISV of the invention, of a Nanobody of the invention,of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

In the context of the present invention, the term “prevention and/ortreatment” not only comprises preventing and/or treating the disease,but also generally comprises preventing the onset of the disease,slowing or reversing the progress of disease, preventing or slowing theonset of one or more symptoms associated with the disease, reducingand/or alleviating one or more symptoms associated with the disease,reducing the severity and/or the duration of the disease and/or of anysymptoms associated therewith and/or preventing a further increase inthe severity of the disease and/or of any symptoms associated therewith,preventing, reducing or reversing any physiological damage caused by thedisease, and generally any pharmacological action that is beneficial tothe patient being treated.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention relates to a method for the prevention and/or treatment ofat least one disease or disorder that is associated with HER3, with itsbiological or pharmacological activity, and/or with the biologicalpathways or signalling in which HER3 is involved, said method comprisingadministering, to a subject in need thereof, a pharmaceutically activeamount of an amino acid sequence of the invention, of an ISV of theinvention, of a Nanobody of the invention, of a polypeptide of theinvention, and/or of a pharmaceutical composition comprising the same.In particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder that can be treatedby modulating HER3, its biological or pharmacological activity, and/orthe biological pathways or signalling in which HER3 is involved, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of an ISV of the invention, of a Nanobody of the invention,of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same. In particular, said pharmaceuticallyeffective amount may be an amount that is sufficient to modulate HER3,its biological or pharmacological activity, and/or the biologicalpathways or signalling in which HER3 is involved; and/or an amount thatprovides a level of the amino acid sequence of the invention, of an ISVof the invention, of a Nanobody of the invention, of a polypeptide ofthe invention in the circulation that is sufficient to modulate HER3,its biological or pharmacological activity, and/or the biologicalpathways or signalling in which HER3 is involved.

The invention furthermore relates to a method for the prevention and/ortreatment of at least one disease or disorder that can be preventedand/or treated by administering an amino acid sequence of the invention,of an ISV of the invention, a Nanobody of the invention or a polypeptideof the invention to a patient, said method comprising administering, toa subject in need thereof, a pharmaceutically active amount of an aminoacid sequence of the invention, of an ISV of the invention, of aNanobody of the invention, of a polypeptide of the invention, and/or ofa pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the preventionand/or treatment of at least one disease or disorder chosen from thegroup consisting of the diseases and disorders listed herein, saidmethod comprising administering, to a subject in need thereof, apharmaceutically active amount of an amino acid sequence of theinvention, of an ISV of the invention, of a Nanobody of the invention,of a polypeptide of the invention, and/or of a pharmaceuticalcomposition comprising the same.

In another aspect, the invention relates to a method for immunotherapy,and in particular for passive immunotherapy, which method comprisesadministering, to a subject suffering from or at risk of the diseasesand disorders mentioned herein, a pharmaceutically active amount of anamino acid sequence of the invention, of an ISV of the invention, of aNanobody of the invention, of a polypeptide of the invention, and/or ofa pharmaceutical composition comprising the same.

In the above methods, the amino acid sequences, ISV's, Nanobodies and/orpolypeptides of the invention and/or the compositions comprising thesame can be administered in any suitable manner, depending on thespecific pharmaceutical formulation or composition to be used. Thus, theamino acid sequences, ISV's, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same can for example beadministered orally, intraperitoneally (e.g. intravenously,subcutaneously, intramuscularly, or via any other route ofadministration that circumvents the gastrointestinal tract),intranasally, transdermally, topically, by means of a suppository, byinhalation, again depending on the specific pharmaceutical formulationor composition to be used. The clinician will be able to select asuitable route of administration and a suitable pharmaceuticalformulation or composition to be used in such administration, dependingon the disease or disorder to be prevented or treated and other factorswell known to the clinician.

The amino acid sequences, ISV's, Nanobodies and/or polypeptides of theinvention and/or the compositions comprising the same are administeredaccording to a regime of treatment that is suitable for preventingand/or treating the disease or disorder to be prevented or treated. Theclinician will generally be able to determine a suitable treatmentregimen, depending on factors such as the disease or disorder to beprevented or treated, the severity of the disease to be treated and/orthe severity of the symptoms thereof, the specific amino acid sequence,ISV, Nanobody or polypeptide of the invention to be used, the specificroute of administration and pharmaceutical formulation or composition tobe used, the age, gender, weight, diet, general condition of thepatient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of oneor more amino acid sequences, ISV's, Nanobodies and/or polypeptides ofthe invention, or of one or more compositions comprising the same, inone or more pharmaceutically effective amounts or doses. The specificamount(s) or doses to administered can be determined by the clinician,again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases anddisorders mentioned herein and depending on the specific disease ordisorder to be treated, the potency of the specific amino acid sequence,ISV, Nanobody and polypeptide of the invention to be used, the specificroute of administration and the specific pharmaceutical formulation orcomposition used, the amino acid sequences, ISV's, Nanobodies andpolypeptides of the invention will generally be administered in anamount between 1 gram and 0.01 milligram per kg body weight per day,preferably between 0.1 gram and 0.01 milligram per kg body weight perday, such as about 0.1, 1, 10, 100 or 1000 milligram per kg body weightper day, either continuously (e.g. by infusion), as a single daily doseor as multiple divided doses during the day. The clinician willgenerally be able to determine a suitable daily dose, depending on thefactors mentioned herein. It will also be clear that in specific cases,the clinician may choose to deviate from these amounts, for example onthe basis of the factors cited above and his expert judgment. Generally,some guidance on the amounts to be administered can be obtained from theamounts usually administered for comparable conventional antibodies orantibody fragments against the same target administered via essentiallythe same route, taking into account however differences inaffinity/avidity, efficacy, biodistribution, half-life and similarfactors well known to the skilled person.

Usually, in the above method, a single amino acid sequence, ISV,Nanobody or polypeptide of the invention will be used. It is howeverwithin the scope of the invention to use two or more amino acidsequences, ISV's, Nanobodies and/or polypeptides of the invention incombination.

The ISV's, Nanobodies, amino acid sequences and polypeptides of theinvention may also be used in combination with one or more furtherpharmaceutically active compounds or principles, i.e. as a combinedtreatment regimen, which may or may not lead to a synergistic effect.Again, the clinician will be able to select such further compounds orprinciples, as well as a suitable combined treatment regimen, based onthe factors cited above and his expert judgement.

In particular, the amino acid sequences, ISV's, Nanobodies andpolypeptides of the invention may be used in combination with otherpharmaceutically active compounds or principles that are or can be usedfor the prevention and/or treatment of the diseases and disorders citedherein, as a result of which a synergistic effect may or may not beobtained. Examples of such compounds and principles, as well as routes,methods and pharmaceutical formulations or compositions foradministering them will be clear to the clinician.

When two or more substances or principles are to be used as part of acombined treatment regimen, they can be administered via the same routeof administration or via different routes of administration, atessentially the same time or at different times (e.g. essentiallysimultaneously, consecutively, or according to an alternating regime).When the substances or principles are to be administered simultaneouslyvia the same route of administration, they may be administered asdifferent pharmaceutical formulations or compositions or part of acombined pharmaceutical formulation or composition, as will be clear tothe skilled person.

Also, when two or more active substances or principles are to be used aspart of a combined treatment regimen, each of the substances orprinciples may be administered in the same amount and according to thesame regimen as used when the compound or principle is used on its own,and such combined use may or may not lead to a synergistic effect.However, when the combined use of the two or more active substances orprinciples leads to a synergistic effect, it may also be possible toreduce the amount of one, more or all of the substances or principles tobe administered, while still achieving the desired therapeutic action.This may for example be useful for avoiding, limiting or reducing anyunwanted side-effects that are associated with the use of one or more ofthe substances or principles when they are used in their usual amounts,while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to theinvention may be determined and/or followed in any manner known per sefor the disease or disorder involved, as will be clear to the clinician.The clinician will also be able, where appropriate and on a case-by-casebasis, to change or modify a particular treatment regimen, so as toachieve the desired therapeutic effect, to avoid, limit or reduceunwanted side-effects, and/or to achieve an appropriate balance betweenachieving the desired therapeutic effect on the one hand and avoiding,limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desiredtherapeutic effect is achieved and/or for as long as the desiredtherapeutic effect is to be maintained. Again, this can be determined bythe clinician.

In another aspect, the invention relates to the use of an amino acidsequence, ISV, Nanobody or polypeptide of the invention in thepreparation of a pharmaceutical composition for prevention and/ortreatment of at least one variety of cancers; and/or for use in one ormore of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is inparticular a mammal, and more in particular a human being. As will beclear to the skilled person, the subject to be treated will inparticular be a person suffering from, or at risk of, the diseases anddisorders mentioned herein.

The invention also relates to the use of an amino acid sequence, ISV,Nanobody or polypeptide of the invention in the preparation of apharmaceutical composition for the prevention and/or treatment of atleast one disease or disorder that can be prevented and/or treated byadministering an amino acid sequence, ISV, Nanobody or polypeptide ofthe invention to a patient.

More in particular, the invention relates to the use of an amino acidsequence, ISV, Nanobody or polypeptide of the invention in thepreparation of a pharmaceutical composition for the prevention and/ortreatment of variety of cancers, and in particular for the preventionand treatment of one or more of the diseases and disorders listedherein.

Again, in such a pharmaceutical composition, the one or more amino acidsequences, ISV's, Nanobodies or polypeptides of the invention may alsobe suitably combined with one or more other active principles, such asthose mentioned herein.

Finally, although the use of the ISV's or Nanobodies of the invention(as defined herein) and of the polypeptides of the invention is muchpreferred, it will be clear that on the basis of the description herein,the skilled person will also be able to design and/or generate, in ananalogous manner, other amino acid sequences and in particular (single)domain antibodies against HER3, as well as polypeptides comprising such(single) domain antibodies.

For example, it will also be clear to the skilled person that it may bepossible to “graft” one or more of the CDR's mentioned above for theNanobodies of the invention onto such (single) domain antibodies orother protein scaffolds, including but not limited to human scaffolds ornon-immunoglobulin scaffolds. Suitable scaffolds and techniques for suchCDR grafting will be clear to the skilled person and are well known inthe art, see for example those mentioned in WO 08/020,079. For example,techniques known per se for grafting mouse or rat CDR's onto humanframeworks and scaffolds can be used in an analogous manner to providechimeric proteins comprising one or more of the CDR's of the Nanobodiesof the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies of the inventionscontain one or more other CDR sequences than the preferred CDR sequencesmentioned above, these CDR sequences can be obtained in any manner knownper se, for example using one or more of the techniques described in WO08/020,079.

Further uses of the amino acid sequences, ISV's, Nanobodies,polypeptides, nucleic acids, genetic constructs and hosts and host cellsof the invention will be clear to the skilled person based on thedisclosure herein. For example, and without limitation, the amino acidsequences of the invention can be linked to a suitable carrier or solidsupport so as to provide a medium than can be used in a manner known perse to purify HER3 from compositions and preparations comprising thesame. Derivatives of the amino acid sequences of the invention thatcomprise a suitable detectable label can also be used as markers todetermine (qualitatively or quantitatively) the presence of HER3 in acomposition or preparation or as a marker to selectively detect thepresence of HER3 on the surface of a cell or tissue (for example, incombination with suitable cell sorting techniques).

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures:

Preferred Aspects:

-   Aspect A-1: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind to HER3.-   Aspect A-2: An immunoglobulin single variable domain according to    aspect A-1, that is in essentially isolated form.-   Aspect A-3: An immunoglobulin single variable domain according to    aspect A-1 or A-2, for administration to a subject, wherein said    immunoglobulin single variable domain does not naturally occur in    said subject.-   Aspect A-4: An immunoglobulin single variable domain that can    specifically bind to HER3 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.    Such an immunoglobulin single variable domain may in particular be    an immunoglobulin single variable domain according to any of the    preceding aspects.-   Aspect A-5: An immunoglobulin single variable domain that can    specifically bind to HER3 with a rate of association (k_(on)-rate)    of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹. Such an    immunoglobulin single variable domain may in particular be an    immunoglobulin single variable domain according to any of the    preceding aspects.-   Aspect A-6: An immunoglobulin single variable domain that can    specifically bind to HER3 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹, preferably between 10⁻² s⁻¹ and 10⁻⁶    s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between    10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹. Such an immunoglobulin single variable domain    may in particular be an immunoglobulin single variable domain    according to any of the preceding aspects.-   Aspect A-7: An immunoglobulin single variable domain that can    specifically bind to HER3 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 1 nM. Such an immunoglobulin single variable domain may    in particular be an immunoglobulin single variable domain according    to any of the preceding aspects.-   Aspect A-8: An immunoglobulin single variable domain according to    any of the preceding aspects, that is a naturally occurring    immunoglobulin single variable domain (from any suitable species) or    a synthetic or semi-synthetic immunoglobulin single variable domain.-   Aspect A-9: An immunoglobulin single variable domain according to    any of the preceding aspects, that comprises an immunoglobulin fold    or that under suitable conditions is capable of forming an    immunoglobulin fold.-   Aspect A-10: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of 4    framework regions (FR1 to FR4 respectively) and 3 complementarity    determining regions (CDR1 to CDR3 respectively).-   Aspect A-11: An immunoglobulin single variable domain according to    any of the preceding aspects, that is an immunoglobulin sequence.-   Aspect A-12: An immunoglobulin single variable domain according to    any of the preceding aspects, that is a naturally occurring    immunoglobulin sequence (from any suitable species) or a synthetic    or semi-synthetic immunoglobulin sequence.-   Aspect A-13: An immunoglobulin single variable domain according to    any of the preceding aspects that is a humanized immunoglobulin    sequence, a camelized immunoglobulin sequence or an immunoglobulin    sequence that has been obtained by techniques such as affinity    maturation.-   Aspect A-14: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a light    chain variable domain sequence (e.g. a VL-sequence); or of a heavy    chain variable domain sequence (e.g. a VH-sequence).-   Aspect A-15: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a heavy    chain variable domain sequence that is derived from a conventional    four-chain antibody or that essentially consists of a heavy chain    variable domain sequence that is derived from a heavy chain    antibody.-   Aspect A-16: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a domain    antibody (or an immunoglobulin single variable domain that is    suitable for use as a domain antibody), of a single domain antibody    (or an immunoglobulin single variable domain that is suitable for    use as a single domain antibody), of a “dAb” (or an immunoglobulin    single variable domain that is suitable for use as a dAb) or of a    Immunoglobulin single variable domain (including but not limited to    a VHH sequence).-   Aspect A-17: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of an    immunoglobulin single variable domain.-   Aspect A-18: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of an    immunoglobulin single variable domain that has preferably one or    more of the amino acid residues at positions 11, 37, 44, 45, 47, 83,    84, 103, 104 and 108 according to the Kabat numbering chosen from    the Hallmark residues mentioned in Table B-2.-   Aspect A-19: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    polypeptide that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 12 to 26,        in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-20: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of an    immunoglobulin single variable domain that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 12 to 26,        in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect A-21: An immunoglobulin single variable domain according to    any of the preceding aspects, that essentially consists of a    humanized or otherwise sequence optimized immunoglobulin single    variable domain.-   Aspect A-22: An immunoglobulin single variable domain according to    any of the preceding aspects, that in addition to the at least one    binding site for binding against/to HER3, contains one or more    further binding sites for binding against/to other antigens,    proteins or targets.-   Aspect B-1: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind HER3, and that    comprises one or more stretches of amino acid residues chosen from    the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 57 to 71;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 57 to 71;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 57 to 71;    -   d) the amino acid sequences of SEQ ID NO's: 87 to 101;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 87 to 101;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 87 to 101;    -   g) the amino acid sequences of SEQ ID NO's: 117 to 131;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 117 to 131;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 117 to 131;    -   or any suitable combination thereof.

Such an immunoglobulin single variable domain may in particular be animmunoglobulin single variable domain according to any of the aspectsA-1 to A-22.

-   Aspect B-2: An immunoglobulin single variable domain according to    aspect B-1, in which at least one of said stretches of amino acid    residues forms part of the antigen binding site for binding    against/to HER3.-   Aspect B-3: An immunoglobulin single variable domain sequence that    is directed against and/or that can specifically bind HER3 and that    comprises two or more stretches of amino acid residues chosen from    the group consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 57 to 71;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 57 to 71;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 57 to 71;    -   d) the amino acid sequences of SEQ ID NO's: 87 to 101;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 87 to 101;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 87 to 101;    -   g) the amino acid sequences of SEQ ID NO's: 117 to 131;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 117 to 131;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 117 to 131;    -   such that (i) when the first stretch of amino acid residues        corresponds to one of the amino acid sequences according to        a), b) or c), the second stretch of amino acid residues        corresponds to one of the amino acid sequences according to d),        e), f), g), h) or i); (ii) when the first stretch of amino acid        residues corresponds to one of the amino acid sequences        according to d), e) or f), the second stretch of amino acid        residues corresponds to one of the amino acid sequences        according to a), b), c), g), h) or i); or (iii) when the first        stretch of amino acid residues corresponds to one of the amino        acid sequences according to g), h) or i), the second stretch of        amino acid residues corresponds to one of the amino acid        sequences according to a), b), c), d), e) or f).    -   Such an immunoglobulin single variable domain may in particular        be an immunoglobulin single variable domain according to any of        the aspects A-1 to A-22, B-1 or B-2.-   Aspect B-4: An immunoglobulin single variable domain according to    aspect B-3, in which the at least two stretches of amino acid    residues forms part of the antigen binding site for binding against    HER3.-   Aspect B-5: An immunoglobulin single variable domain sequence that    is directed against and/or that can specifically bind HER3 and that    comprises three or more stretches of amino acid residues, in which    the first stretch of amino acid residues is chosen from the group    consisting of:    -   a) the amino acid sequences of SEQ ID NO's: 57 to 71;    -   b) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 57 to 71;    -   c) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 57 to 71;    -   the second stretch of amino acid residues is chosen from the        group consisting of:    -   d) the amino acid sequences of SEQ ID NO's: 87 to 101;    -   e) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 87 to 101;    -   f) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 87 to 101;    -   and the third stretch of amino acid residues is chosen from the        group consisting of:    -   g) the amino acid sequences of SEQ ID NO's: 117 to 131;    -   h) amino acid sequences that have at least 80% amino acid        identity with at least one of the amino acid sequences of SEQ ID        NO's: 117 to 131;    -   i) amino acid sequences that have 3, 2, or 1 amino acid        difference with at least one of the amino acid sequences of SEQ        ID NO's: 117 to 131.    -   Such an immunoglobulin single variable domain may in particular        be an immunoglobulin single variable domain according to any of        the aspects A-1 to A-22 and/or B-1 to B-4.-   Aspect B-6: An immunoglobulin single variable domain according to    aspect B-5, in which the at least three stretches of amino acid    residues forms part of the antigen binding site for binding    against/to HER3.-   Aspect B-7: An immunoglobulin single variable domain that is    directed against and/or that can specifically bind HER3 in which the    CDR sequences of said immunoglobulin single variable domain have at    least 70% amino acid identity, preferably at least 80% amino acid    identity, more preferably at least 90% amino acid identity, such as    95% amino acid identity or more or even essentially 100% amino acid    identity with the CDR sequences of at least one of the    immunoglobulin single variable domains of SEQ ID NO's: 12 to 26.    Such an immunoglobulin single variable domain may in particular be    an immunoglobulin single variable domain according to any of the    aspects A-1 to A-22 and/or B-1 to B-6.-   Aspect C-1: An immunoglobulin single variable domain that is    directed against HER3 and that cross-blocks the binding of at least    one of the immunoglobulin single variable domains of SEQ ID NO's: 12    to 26 to HER3. Such an immunoglobulin single variable domain may in    particular be an immunoglobulin single variable domain according to    any of the aspects A-1 to A-22 and/or according to aspects B-1 to    B-7. Also, preferably, such an immunoglobulin single variable domain    is able to specifically bind to HER3.-   Aspect C-2: An immunoglobulin single variable domain that is    directed against HER3 and that is cross-blocked from binding to HER3    by at least one of the immunoglobulin single variable domains of SEQ    ID NO's: 12 to 26. Such an immunoglobulin single variable domain may    in particular be an immunoglobulin single variable domain according    to any of the aspects A-1 to A-22 and/or according to aspects B-1 to    B-7. Also, preferably, such an immunoglobulin single variable domain    is able to specifically bind to HER3.-   Aspect C-3: An immunoglobulin single variable domain according to    any of aspects C-1 or C-2, wherein the ability of said    immunoglobulin single variable domain to cross-block or to be    cross-blocked is detected in a FACS competition assay, e.g. as    described in the experimental part.-   Aspect C-4: An immunoglobulin single variable domain according to    any of aspects C-1 to C-3, wherein the ability of said    immunoglobulin single variable domain to cross-block or to be    cross-blocked is detected in an ELISA assay.-   Aspect D-1: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7 or C-1 to C-4, that is in essentially    isolated form.-   Aspect D-2: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, and/or D1 for administration    to a subject, wherein said immunoglobulin single variable domain    does not naturally occur in said subject.-   Aspect D-3: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, and/or D1 to D-2 that can    specifically bind to HER3 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.-   Aspect D-4: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, and/or D-1 to D-3 that can    specifically bind to HER3 with a rate of association (k_(on)-rate)    of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹ s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect D-5: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, and/or D-1 to D-4 that can    specifically bind to HER3 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect D-6: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, and/or D-1 to D-5 that can    specifically bind to HER3 with an affinity less than 500 nM,    preferably less than 100 nM, more preferably less than 10 nM, such    as less than 1 nM.    -   The immunoglobulin single variable domains according to aspects        D-1 to D-6 may in particular be an immunoglobulin single        variable domain according to any of the aspects A-1 to A-22.-   Aspect E-1: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4 and/or D1 to D-6, that is a    naturally occurring immunoglobulin single variable domain (from any    suitable species) or a synthetic or semi-synthetic immunoglobulin    single variable domain.-   Aspect E-2: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 that    comprises an immunoglobulin fold or that under suitable conditions    is capable of forming an immunoglobulin fold.-   Aspect E-3: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or D-1 or D-2,    that is an immunoglobulin sequence.-   Aspect E-4: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-3,    that is a naturally occurring immunoglobulin sequence (from any    suitable species) or a synthetic or semi-synthetic immunoglobulin    sequence.-   Aspect E-5: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-4    that is a humanized immunoglobulin sequence, a camelized    immunoglobulin sequence or an immunoglobulin sequence that has been    obtained by techniques such as affinity maturation.-   Aspect E-6: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-5    that essentially consists of a light chain variable domain sequence    (e.g. a V_(L)-sequence); or of a heavy chain variable domain    sequence (e.g. a V_(H)-sequence).-   Aspect E-7: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-6,    that essentially consists of a heavy chain variable domain sequence    that is derived from a conventional four-chain antibody or that    essentially consist of a heavy chain variable domain sequence that    is derived from heavy chain antibody.-   Aspect E-8: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-7,    that essentially consists of a domain antibody (or an immunoglobulin    single variable domain that is suitable for use as a domain    antibody), of a single domain antibody (or an immunoglobulin single    variable domain that is suitable for use as a single domain    antibody), of a “dAb” (or an immunoglobulin single variable domain    that is suitable for use as a dAb) or of an immunoglobulin single    variable domain (including but not limited to a V_(HH) sequence).-   Aspect E-9: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-8    that essentially consists of a VHH or engineered VHH.-   Aspect E-10: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-9    that essentially consists of a VHH or engineered VHH that has    preferably one or more of the amino acid residues at positions 11,    37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2.-   Aspect E-11: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to    E-10, that essentially consists of a VHH or engineered VHH that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 12 to 26,        in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect E-12: An immunoglobulin single variable domain according to    any of aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to E-11    that essentially consists of a VHH or engineered VHH.-   Aspect E-13: An immunoglobulin single variable domain according to    any of the aspects B-1 to B-7, C-1 to C-4, D1 to D-6, and/or E-1 to    E-11, that in addition to the at least one stretch of amino acid    residues or binding site for binding formed by the CDR sequences,    contains one or more further binding sites for binding against other    antigens, proteins or targets.    -   The immunoglobulin single variable domains according to aspects        E-1 to E-13 may in particular be an immunoglobulin single        variable domain according to any of the aspects A-1 to A-22.-   Aspect F-1: An immunoglobulin single variable domain that    essentially consists of 4 framework regions (FR1 to FR4,    respectively) and 3 complementarity determining regions (CDR1 to    CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID            NO's: 57 to 71;        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   and/or    -   CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domains of SEQ ID            NO's: 87 to 101;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   and/or    -   CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID            NO's: 117 to 131;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131.        -   Such an immunoglobulin single variable domain is preferably            directed against HER3 and/or an immunoglobulin single            variable domain that can specifically bind to HER3. Also,            such an immunoglobulin single variable domain is preferably            an immunoglobulin single variable domain according to any of            the aspects A-1 to A-22, C-1 to C-4, D1 to D-6 and/or E-1 to            E-13.-   Aspect F-2: An immunoglobulin single variable domain that    essentially consists of 4 framework regions (FR1 to FR4,    respectively) and 3 complementarity determining regions (CDR1 to    CDR3, respectively), in which:    -   CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID            NO's: 57 to 71;        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   and    -   CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domains of SEQ ID            NO's: 87 to 101;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   and    -   CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID            NO's: 117 to 131;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131.        -   Such an immunoglobulin single variable domain is preferably            directed against HER3 and/or an immunoglobulin single            variable domain that can specifically bind to HER3. Also,            such an immunoglobulin single variable domain is preferably            an immunoglobulin single variable domain according to any of            the aspects A-1 to A-22, C-1 to C-4, D1 to D-6 and/or E-1 to            E-13.-   Aspect F-3: An immunoglobulin single variable domain according to    any of aspects F-1 and F-2, in which the CDR sequences of said    immunoglobulin single variable domain have at least 70% amino acid    identity, preferably at least 80% amino acid identity, more    preferably at least 90% amino acid identity, such as 95% amino acid    identity or more or even essentially 100% amino acid identity with    the CDR sequences of at least one of the immunoglobulin single    variable domains of SEQ ID NO's: 12 to 26.    -   Such an immunoglobulin single variable domain is preferably        directed against HER3 and/or an immunoglobulin single variable        domain that can specifically bind to HER3. Also, such an        immunoglobulin single variable domain is preferably an        immunoglobulin single variable domain according to any of the        aspects A-1 to A-22, C-1 to C-4, D1 to D-6 and/or E-1 to E-13.-   Aspect F-4: An immunoglobulin single variable domain according to    any of aspects F-1 to F-3 that is directed against HER3 and that    cross-blocks the binding of at least one of the immunoglobulin    single variable domains according to any of aspects the    immunoglobulin single variable domains of SEQ ID NO's: 12 to 26.-   Aspect F-5: An immunoglobulin single variable domain according to    any of aspects F-1 to F-3 that is directed against HER3 and that is    cross-blocked from binding to HER3 by at least one of the    immunoglobulin single variable domains of SEQ ID NO's: 12 to 26.-   Aspect F-6: Immunoglobulin single variable domain according to any    of aspects F-4 or F-5 wherein the ability of said immunoglobulin    single variable domain to cross-block or to be cross-blocked is    detected in a FACS competition assay as e.g. shown in the    experimental part.-   Aspect F-7: Immunoglobulin single variable domain according to any    of aspects F4 or F-5 wherein the ability of said immunoglobulin    single variable domain to cross-block or to be cross-blocked is    detected in an ELISA assay.-   Aspect F-8: An immunoglobulin single variable domain according to    any of aspects F-1 to F-7, that is in essentially isolated form.-   Aspect F-9: An immunoglobulin single variable domain according to    any of aspects F-1 to F-8, for administration to a subject, wherein    said an immunoglobulin single variable domain does not naturally    occur in said subject.-   Aspect F-10: An immunoglobulin single variable domain according to    any of aspects F-1 to F-9, that can specifically bind to HER3 with a    dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or less,    and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more preferably    10⁻⁸ to 10⁻¹² moles/liter.-   Aspect F-11: An immunoglobulin single variable domain according to    any of aspects F-1 to F-10, that can specifically bind to HER3 with    a rate of association (k_(on)-rate) of between 10² M⁻¹s⁻¹ to about    10⁷ M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more    preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect F-12: An immunoglobulin single variable domain according to    any of aspects F-1 to F-11, that can specifically bind to HER3 with    a rate of dissociation (k_(off) rate) between 1 s⁻¹ and 10⁻⁶ s⁻¹    preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between    10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect F-13: An immunoglobulin single variable domain according to    any of aspects F-1 to F-12, that can specifically bind to HER3 with    an affinity less than 500 nM, preferably less than 200 nM, more    preferably less than 10 nM, such as less than 1 nM.-   Aspect F-14: An immunoglobulin single variable domain according to    any of aspects F-1 to F-13, that is a naturally occurring    immunoglobulin single variable domain (from any suitable species) or    a synthetic or semi-synthetic immunoglobulin single variable domain.-   Aspect F-15: An immunoglobulin single variable domain according to    any of aspects F-1 to F-14, that comprises an immunoglobulin fold or    that under suitable conditions is capable of forming an    immunoglobulin fold.-   Aspect F-16: An immunoglobulin single variable domain according to    any of aspects F-1 to F-15, that is an immunoglobulin sequence.-   Aspect F-17: An immunoglobulin single variable domain according to    any of aspects F-1 to F-16, that is a naturally occurring    immunoglobulin sequence (from any suitable species) or a synthetic    or semi-synthetic immunoglobulin sequence.-   Aspect F-18: An immunoglobulin single variable domain according to    any of aspects F-1 to F-17, that is a humanized immunoglobulin    sequence, a camelized immunoglobulin sequence or an immunoglobulin    sequence that has been obtained by techniques such as affinity    maturation.-   Aspect F-19: An immunoglobulin single variable domain according to    any of aspects F-1 to F-19, that essentially consists of a light    chain variable domain sequence (e.g. a V_(L)-sequence); or of a    heavy chain variable domain sequence (e.g. a V_(H)-sequence).-   Aspect F-20: An immunoglobulin single variable domain according to    any of aspects F-1 to F-19, that essentially consists of a heavy    chain variable domain sequence that is derived from a conventional    four-chain antibody or that essentially consist of a heavy chain    variable domain sequence that is derived from heavy chain antibody.-   Aspect F-21: An immunoglobulin single variable domain according to    any of aspects F-1 to F-20, that essentially consists of a domain    antibody (or an immunoglobulin single variable domain that is    suitable for use as a domain antibody), of a single domain antibody    (or an immunoglobulin single variable domain that is suitable for    use as a single domain antibody), of a “dAb” (or an immunoglobulin    single variable domain that is suitable for use as a dAb) or of a    VHH or engineered VHH.-   Aspect F-22: An immunoglobulin single variable domain according to    any of aspects F-1 to F-21, that essentially consists of a VHH or    engineered VHH.-   Aspect F-23: An immunoglobulin single variable domain according to    any of aspects F-1 to F-22, that essentially consists of a VHH or    engineered VHH that has preferably one or more of the amino acid    residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108    according to the Kabat numbering are chosen from the Hallmark    residues mentioned in Table B-2.-   Aspect F-24: An immunoglobulin single variable domain according to    any of aspects F-1 to F-23, that essentially consists of an    immunoglobulin single variable domain that    -   i) has at least 80% amino acid identity with at least one of the        immunoglobulin single variable domains of SEQ ID NO's: 12 to 26,        in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect F-25: An immunoglobulin single variable domain according to    any of aspects F-1 to F-24, that essentially consists of a sequence    optimized VHH.-   Aspect G-1: An immunoglobulin single variable domain according to    any of the preceding aspects, that in addition to the at least one    binding site for binding formed by the CDR sequences, contains one    or more further binding sites for binding against another antigen,    protein or target.-   Aspect H-1: VHH that is directed against and/or that can    specifically bind to HER3.-   Aspect H-2: VHH according to aspect H-1, that is in essentially    isolated form.-   Aspect H-3: VHH according to any of aspects H-1 to H-2, that can    specifically bind to HER3 with a dissociation constant (K_(D)) of    10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²    moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter.-   Aspect H-4: VHH according to any of aspects H-1 to H-3, that can    specifically bind to HER3 with a rate of association (k_(on)-rate)    of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, preferably between 10³    M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between 10⁴ M⁻¹s⁻¹ and 10⁷    M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹.-   Aspect H-5: VHH according to any of aspects H-1 to H-4, that can    specifically bind to HER3 with a rate of dissociation (k_(off) rate)    between 1 s⁻¹ and 10⁻⁶ s⁻¹ preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,    more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹, such as between 10⁻⁴    s⁻¹ and 10⁻⁶ s⁻¹.-   Aspect H-6: VHH according to any of aspects H-1 to H-5, that can    specifically bind to HER3 with an affinity less than 500 nM,    preferably less than 200 nM, more preferably less than 10 nM, such    as less than 500 pM.-   Aspect H-7: VHH according to any of aspects H-1 to H-6, that is a    naturally occurring VHH (from e.g. a llama) or a synthetic or    semi-synthetic VHH.-   Aspect H-8: VHH according to any of aspects to H-1 to H-7, that is a    V_(HH) sequence, a partially humanized V_(HH) sequence, a fully    humanized V_(HH) sequence, a camelized heavy chain variable domain    or a VHH that has been obtained by techniques such as affinity    maturation.-   Aspect H-9: VHH according to any of aspects H-1 to H-8, that has    preferably one or more of the amino acid residues at positions 11,    37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat    numbering are chosen from the Hallmark residues mentioned in Table    B-2.-   Aspect H-10: VHH according to any of aspects H-1 to H-9, that    -   i) has at least 80% amino acid identity with at least one of the        An immunoglobulin single variable domains of SEQ ID NO's: 12 to        26, in which for the purposes of determining the degree of amino        acid identity, the amino acid residues that form the CDR        sequences are disregarded;    -   and in which:    -   ii) preferably one or more of the amino acid residues at        positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according        to the Kabat numbering are chosen from the Hallmark residues        mentioned in Table B-2.-   Aspect H-11: VHH according to any of aspects H-1 to H-10, in which:    -   CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID            NO's: 57 to 71;        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   and/or    -   CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domains of SEQ ID            NO's: 87 to 101;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   and/or    -   CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID            NO's: 117 to 131;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131.-   Aspect H-12: VHH according to any of aspects H-1 to H-11, in which:    -   CDR1 is chosen from the group consisting of:        -   a) the immunoglobulin single variable domains of SEQ ID            NO's: 57 to 71;        -   b) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   c) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 57 to            71;        -   and    -   CDR2 is chosen from the group consisting of:        -   d) the immunoglobulin single variable domains of SEQ ID            NO's: 87 to 101;        -   e) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   f) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 87 to            101;        -   and    -   CDR3 is chosen from the group consisting of:        -   g) the immunoglobulin single variable domains of SEQ ID            NO's: 117 to 131;        -   h) immunoglobulin single variable domains that have at least            80% amino acid identity with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131;        -   i) immunoglobulin single variable domains that have 3, 2, or            1 amino acid difference with at least one of the            immunoglobulin single variable domains of SEQ ID NO's: 117            to 131.-   Aspect H-13: VHH according to any of aspects H-1 to H-12, in which    the CDR sequences have at least 70% amino acid identity, preferably    at least 80% amino acid identity, more preferably at least 90% amino    acid identity, such as 95% amino acid identity or more or even    essentially 100% amino acid identity with the CDR sequences of at    least one of the immunoglobulin single variable domains of SEQ ID    NO's: 12 to 26.-   Aspect H-14: VHH according to any of aspects H-1 to H-13, which is a    partially humanized VHH.-   Aspect H-15: VHH according to any of aspects H-1 to H-14, which is a    fully humanized VHH.-   Aspect H-16: VHH according to any of aspects H-1 to H-15, that is    chosen from the group consisting of SEQ ID NO's: 12 to 26 or from    the group consisting of from immunoglobulin single variable domains    that have more than 80%, preferably more than 90%, more preferably    more than 95%, such as 99% or more sequence identity (as defined    herein) with at least one of the immunoglobulin single variable    domains of SEQ ID NO's: 12 to 26.-   Aspect H-17: VHH according to any of aspects H-1 to H-16, which is a    humanized VHH.-   Aspect H-18: VHH according to any of aspects H-1 to H-17, that is    chosen from the group consisting of SEQ ID NO's: 12 to 26.-   Aspect H-19: VHH directed against HER3 that cross-blocks the binding    of at least one of the immunoglobulin single variable domains of SEQ    ID NO's: 12 to 26 to HER3.-   Aspect H-20: VHH directed against HER3 that is cross-blocked from    binding to HER3 by at least one of the immunoglobulin single    variable domains of SEQ ID NO's: 12 to 26.-   Aspect H-21: VHH according to any of aspects H-19 or H-20 wherein    the ability of said VHH to cross-block or to be cross-blocked is    detected in a FACS competition assay, e.g. as described in the    experimental part.-   Aspect H-22: VHH according to any of aspects H-19 to H-21 wherein    the ability of said VHH to cross-block or to be cross-blocked is    detected in an ELISA assay.-   Aspect K-1: Polypeptide that comprises or essentially consists of    one or more immunoglobulin single variable domains according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25, G-1 and/or one or more VHH according to any of    aspects H-1 to H-22, and optionally further comprises one or more    peptidic linkers and/or one or more other groups, residues, moieties    or binding units.-   Aspect K-2: Polypeptide according to aspect K-1, in which said one    or more binding units are immunoglobulin sequences, and in    particular ISV's.-   Aspect K-3: Polypeptide according to any of aspects K-1 or K-2, in    which said one or more other groups, residues, moieties or binding    units are chosen from the group consisting of domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a domain antibody, single domain antibodies, immunoglobulin single    variable domains that are suitable for use as a single domain    antibody, “dAb”'s, immunoglobulin single variable domains that are    suitable for use as a dAb, or VHHs.-   Aspect K-4: Polypeptide according to any of aspects K-1 to K-3, in    which said one or more immunoglobulin single variable domains of the    invention are immunoglobulin sequences.-   Aspect K-5: Polypeptide according to any of aspects K-1 to K-4, in    which said one or more immunoglobulin single variable domains of the    invention are chosen from the group consisting of domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a domain antibody, single domain antibodies, immunoglobulin single    variable domains that are suitable for use as a single domain    antibody, “dAb”'s, immunoglobulin single variable domains that are    suitable for use as a dAb, or VHHs.-   Aspect K-6: Polypeptide according to any of aspects K-1 to K-5, that    comprises or essentially consists of one or more Nanobodies    according to any of aspects H-1 to H-22 and in which said one or    more other binding units are Nanobodies.-   Aspect K-7: Polypeptide according to any of aspects K-1 to K-6,    wherein at least one binding unit is a multivalent construct.-   Aspect K-8: Polypeptide according to any of aspects K-1 to K-7,    wherein at least one binding unit is a multiparatopic construct.-   Aspect K-9: Polypeptide according to any of aspects K-1 to K-8,    wherein at least one binding unit is a multispecific construct.-   Aspect K-10: Polypeptide according to any of aspects K-1 to K-9,    which has an increased half-life, compared to the corresponding    immunoglobulin single variable domain according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1 per se or VHH according to any of aspects H-1 to H-22    per se, respectively.-   Aspect K-11: Polypeptide according to aspect K-10, in which said one    or more other binding units provide the polypeptide with increased    half-life, compared to the corresponding immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or    VHH according to any of aspects H-1 to H-22 per se, respectively.-   Aspect K-12: Polypeptide according to aspect K-10 or K-11, in which    said one or more other binding units that provide the polypeptide    with increased half-life is chosen from the group consisting of    serum proteins or fragments thereof, binding units that can bind to    serum proteins, an Fc portion, and small proteins or peptides that    can bind to serum proteins.-   Aspect K-13: Polypeptide according to any of aspects K-10 to K-12,    in which said one or more other binding units that provide the    polypeptide with increased half-life is chosen from the group    consisting of human serum albumin or fragments thereof.-   Aspect K-14: Polypeptide according to any of aspect K-10 to K-13, in    which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of binding units that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-15: Polypeptide according to any of aspects K-10 to K-14,    in which said one or more other binding units that provides the    polypeptide with increased half-life are chosen from the group    consisting of domain antibodies, immunoglobulin single variable    domains that are suitable for use as a domain antibody, single    domain antibodies, immunoglobulin single variable domains that are    suitable for use as a single domain antibody, “dAb”'s,    immunoglobulin single variable domains that are suitable for use as    a dAb, or VHHs that can bind to serum albumin (such as human serum    albumin) or a serum immunoglobulin (such as IgG).-   Aspect K-16: Polypeptide according to aspect K-10 to K-15, in which    said one or more other binding units that provides the polypeptide    with increased half-life is a VHH that can bind to serum albumin    (such as human serum albumin) or a serum immunoglobulin (such as    IgG).-   Aspect K-17: Polypeptide according to any of aspects K-10 to K-16,    that has a serum half-life that is at least 1.5 times, preferably at    least 2 times, such as at least 5 times, for example at least 10    times or more than 20 times, greater than the half-life of the    corresponding immunoglobulin single variable domain according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 per se or a VHH according to any of aspects    H-1 to H-22 per se, respectively.-   Aspect K-18: Polypeptide according to any of aspects K-10 to K-17,    that has a serum half-life that is increased with more than 1 hours,    preferably more than 2 hours, more preferably more than 6 hours,    such as more than 12 hours, or even more than 24, 48 or 72 hours,    compared to the corresponding immunoglobulin single variable domain    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or a VHH according to    any of aspects H-1 to H-22 per se, respectively.-   Aspect K-19: Polypeptide according to any of aspects K-1 to K-18,    that has a serum half-life in human of at least about 12 hours,    preferably at least 24 hours, more preferably at least 48 hours,    even more preferably at least 72 hours or more; for example, of at    least 5 days (such as about 5 to 10 days), preferably at least 9    days (such as about 9 to 14 days), more preferably at least about 10    days (such as about 10 to 15 days), or at least about 11 days (such    as about 11 to 16 days), more preferably at least about 12 days    (such as about 12 to 18 days or more), or more than 14 days (such as    about 14 to 19 days).-   Aspect L-1: Compound or construct, that comprises or essentially    consists of one or more immunoglobulin single variable domains    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1 and/or one or more VHHs    according to any of aspects H-1 to H-22, and optionally further    comprises one or more other groups, residues, moieties or binding    units, optionally linked via one or more linkers.-   Aspect L-2: Compound or construct according to aspects L-1, in which    said one or more other groups, residues, moieties or binding units    are immunoglobulin single variable domains.-   Aspect L-3: Compound or construct according to aspect L-1 or L-2, in    which said one or more linkers, if present, are one or more    immunoglobulin single variable domains.-   Aspect L-4: Compound or construct according to any of aspects L-1 to    L-3, in which said one or more other groups, residues, moieties or    binding units are immunoglobulin sequences.-   Aspect L-5: Compound or construct according to any of aspects L-1 to    L-4, in which said one or more other groups, residues, moieties or    binding units are chosen from the group consisting of domain    antibodies, immunoglobulin single variable domains that are suitable    for use as a domain antibody, single domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a single domain antibody, “dAb”'s, immunoglobulin single variable    domains that are suitable for use as a dAb, or VHHs.-   Aspect L-6: Compound or construct according to any of aspects L-1 to    L-5, in which said one or more immunoglobulin single variable    domains of the invention are immunoglobulin sequences.-   Aspect L-7: Compound or construct according to any of aspects L-1 to    L-6, in which said one or more immunoglobulin single variable    domains of the invention are chosen from the group consisting of    domain antibodies, immunoglobulin single variable domains that are    suitable for use as a domain antibody, single domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a single domain antibody, “dAb”'s, immunoglobulin single variable    domains that are suitable for use as a dAb, or VHHs.-   Aspect L-8: Compound or construct, that comprises or essentially    consists of one or more VHH's or Nanobodies according to any of    aspects H-1 to H-22 and in which said one or more other groups,    residues, moieties or binding units are VHHs.-   Aspect L-9: Compound or construct according to any of aspects L-1 to    L-8, which is a multivalent construct.-   Aspect L-10: Compound or construct according to any of aspects L-1    to L-9, which is a multispecific construct.-   Aspect L-11: Compound or construct according to any of aspects L-1    to L-10, which has an increased half-life, compared to the    corresponding immunoglobulin single variable domain according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1 per se or VHH according to any of aspects    H-1 to H-22 per se, respectively.-   Aspect L-12: Compound or construct according to aspect L-1 to L-11,    in which said one or more other groups, residues, moieties or    binding units provide the compound or construct with increased    half-life, compared to the corresponding immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or    VHH according to any of aspects H-1 to H-22 per se, respectively.-   Aspect L-13: Compound or construct according to aspect L-12, in    which said one or more other groups, residues, moieties or binding    units that provide the compound or construct with increased    half-life is chosen from the group consisting of serum proteins or    fragments thereof, binding units that can bind to serum proteins, an    Fc portion, and small proteins or peptides that can bind to serum    proteins.-   Aspect L-14: Compound or construct according to aspect L-12 or L-13,    in which said one or more other groups, residues, moieties or    binding units that provide the compound or construct with increased    half-life is chosen from the group consisting of human serum albumin    or fragments thereof.-   Aspect L-15: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of binding    units that can bind to serum albumin (such as human serum albumin)    or a serum immunoglobulin (such as IgG).-   Aspect L-16: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life are chosen from the group consisting of domain    antibodies, immunoglobulin single variable domains that are suitable    for use as a domain antibody, single domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a single domain antibody, “dAb”'s, immunoglobulin single variable    domains that are suitable for use as a dAb, or VHHs that can bind to    serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).-   Aspect L-17: Compound or construct according to any of aspects L-12    to L-14, in which said one or more other groups, residues, moieties    or binding units that provides the compound or construct with    increased half-life is a VHH that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).-   Aspect L-18: Compound or construct according to any of aspects L-12    to L-17, that has a serum half-life that is at least 1.5 times,    preferably at least 2 times, such as at least 5 times, for example    at least 10 times or more than 20 times, greater than the half-life    of the corresponding immunoglobulin single variable domain according    to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6,    E-1 to E-13, F-1 to F-25 or G-1 per se or VHH according to any of    aspects H-1 to H-22 per se, respectively.-   Aspect L-19: Compound or construct according to any of aspects L-12    to L-18, that has a serum half-life that is increased with more than    1 hours, preferably more than 2 hours, more preferably more than 6    hours, such as more than 12 hours, or even more than 24, 48 or 72    hours, compared to the corresponding immunoglobulin single variable    domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1 per se or VHH    according to any of aspects H-1 to H-22 per se, respectively.-   Aspect L-20: Compound or construct according to any of aspects L-12    to L-19, that has a serum half-life in human of at least about 12    hours, preferably at least 24 hours, more preferably at least 48    hours, even more preferably at least 72 hours or more; for example,    of at least 5 days (such as about 5 to 10 days), preferably at least    9 days (such as about 9 to 14 days), more preferably at least about    10 days (such as about 10 to 15 days), or at least about 11 days    (such as about 11 to 16 days), more preferably at least about 12    days (such as about 12 to 18 days or more), or more than 14 days    (such as about 14 to 19 days).-   Aspect L-21: Monovalent construct, comprising or essentially    consisting of one immunoglobulin single variable domain according to    any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1    to E-13, F-1 to F-25 or G-1 and/or one VHH according to any of    aspects H-1 to H-22.-   Aspect L-22: Monovalent construct according to aspect L-21, in which    said immunoglobulin single variable domain of the invention is    chosen from the group consisting of domain antibodies,    immunoglobulin single variable domains that are suitable for use as    a domain antibody, single domain antibodies, immunoglobulin single    variable domains that are suitable for use as a single domain    antibody, “dAb”'s, immunoglobulin single variable domains that are    suitable for use as a dAb, or VHHs.-   Aspect L-23: Monovalent construct, comprising or essentially    consisting of one VHH according to any of aspects H-1 to H-22.-   Aspect M-1: Nucleic acid or nucleotide sequence, that encodes an    immunoglobulin single variable domain according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1, a VHH according to any of aspects H-1 to H-22, a    compound or construct according to any of aspects that is such that    it can be obtained by expression of a nucleic acid or nucleotide    sequence encoding the same; e.g. a nucleic acid or nucleotide    sequence that has at least 70% sequence identity, preferably at    least 80% sequence identity, more preferably at least 90% sequence    identity, such as 95% sequence identity or more or even essentially    100% sequence identity with the sequences of at least one of the    nucleic acid or nucleotide sequence of SEQ ID NO's: 27 to 41.-   Aspect M-2: Nucleic acid or nucleotide sequence according to aspect    M-1, that is in the form of a genetic construct.-   Aspect N-1: Host or host cell that expresses, or that under suitable    circumstances is capable of expressing, an immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a VHH    according to any of aspects H-1 to H-22, a polypeptide according to    any of aspects K-1 to K-19, a compound or construct according to any    of aspects L-1 to L-21 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same, or a monovalent construct according to any of aspects L-22 or    L-23; and/or that comprises a nucleic acid or nucleotide sequence    according to aspect M-1 or a genetic construct according to aspect    M-2.-   Aspect O-1: Composition comprising at least one immunoglobulin    single variable domain according to any of aspects A-1 to A-22, B-1    to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a    VHH according to any of aspects H-1 to H-22, a polypeptide according    to any of aspects K-1 to K-19, a compound or construct according to    any of aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23, or a nucleic acid or nucleotide sequence    according to aspects M-1 or M-2.-   Aspect O-2: Composition according to aspect O-1, which is a    pharmaceutical composition.-   Aspect O-3: Composition according to aspect O-2, which is a    pharmaceutical composition, that further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and that optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.-   Aspect P-1: Method for producing an immunoglobulin single variable    domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a VHH according to    any of aspects H-1 to H-22, a polypeptide according to any of    aspects K-1 to K-19, a compound or construct according to any of    aspects L-1 to L-21 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same, or a monovalent construct according to any of aspects L-22 or    L-23, said method at least comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid or nucleotide        sequence according to aspect M-1, or a genetic construct        according to aspect M-2;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin single variable        domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1        to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a VHH        according to any of aspects H-1 to H-22, a polypeptide according        to any of aspects K-1 to K-19, a compound or construct according        to any of aspects L-1 to L-21, or a monovalent construct        according to any of aspects L-22 or L-23 thus obtained.-   Aspect P-2: Method for producing an immunoglobulin single variable    domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to    C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, a VHH according to    any of aspects H-1 to H-22, a polypeptide according to any of    aspects K-1 to K-19, a compound or construct according to any of    aspects L-1 to L-21 that is such that it can be obtained by    expression of a nucleic acid or nucleotide sequence encoding the    same, or a monovalent construct according to any of aspects L-22 or    L-23, said method at least comprising the steps of:    -   a) cultivating and/or maintaining a host or host cell according        to aspect N1, under conditions that are such that said host or        host cell expresses and/or produces at least one immunoglobulin        single variable domain according to any of aspects A-1 to A-22,        B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or        G-1, a VHH according to any of aspects H-1 to H-22, a        polypeptide according to any of aspects K-1 to K-19, a compound        or construct according to any of aspects L-1 to L-21, or        monovalent construct according to any of aspects L-22 or L-23;    -   optionally followed by:    -   b) isolating and/or purifying the immunoglobulin single variable        domain according to any of aspects A-1 to A-22, B-1 to B-7, C-1        to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, VHH        according to any of aspects H-1 to H-22, a polypeptide according        to any of aspects K-1 to K-19, a compound or construct according        to any of aspects L-1 to L-21, or monovalent construct according        to any of aspects L-22 or L-23 thus obtained.-   Aspect Q-1: Method for screening immunoglobulin single variable    domains directed against HER3 that comprises at least the steps of:    -   a) providing a set, collection or library of nucleic acid        sequences encoding immunoglobulin single variable domains;    -   b) screening said set, collection or library of nucleic acid        sequences for nucleic acid sequences that encode an        immunoglobulin single variable domain that can bind to and/or        has affinity for HER3 and that is cross-blocked or is cross        blocking a Immunoglobulin single variable domain of the        invention, e.g. SEQ ID NO: 12 to 26 (Table-A-1), or a humanized        Immunoglobulin single variable domain of the invention, or a        polypeptide or construct of the invention, e.g. SEQ ID NO: 147        to 327, more preferably HER3MS00135 (SEQ ID NO:282), HER3MS00212        (SEQ ID NO:319) or HER3MS00215 (SEQ ID NO:322). (see Table A-2);        and    -   c) isolating said nucleic acid sequence, followed by expressing        said immunoglobulin single variable domain.-   Aspect R-1: Method for the prevention and/or treatment of at least    one variety of cancers, said method comprising administering, to a    subject in need thereof, a pharmaceutically active amount of at    least one immunoglobulin single variable domain according to any of    aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, VHH according to any of aspects H-1 to    H-22, polypeptide according to any of aspects K-1 to K-19, compound    or construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3.-   Aspect R-2: Method for the prevention and/or treatment of at least    one disease or disorder that is associated with HER3, with its    biological or pharmacological activity, and/or with the biological    pathways or signalling in which HER3 is involved, said method    comprising administering, to a subject in need thereof, a    pharmaceutically active amount of at least one immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, VHH    according to any of aspects H-1 to H-22, polypeptide according to    any of aspects K-1 to K-19, compound or construct according to any    of aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23; or composition according to aspect O-2 or O-3.-   Aspect R-3: Method for the prevention and/or treatment of at least    one disease or disorder that can be prevented and/or treated by    administering, to a subject in need thereof, at least one    immunoglobulin single variable domain according to any of aspects    A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to    F-25 or G-1, VHH according to any of aspects H-1 to H-22,    polypeptide according to any of aspects K-1 to K-19, compound or    construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one at least one immunoglobulin single    variable domain according to any of aspects A-1 to A-22, B-1 to B-7,    C-1 to C-4, D-1 to D-6, E-1 to E-13, F-1 to F-25 or G-1, VHH    according to any of aspects H-1 to H-22, polypeptide according to    any of aspects K-1 to K-19, compound or construct according to any    of aspects L-1 to L-21, monovalent construct according to any of    aspects L-22 or L-23; or composition according to aspect O-2 or O-3.-   Aspect R-4: Method for immunotherapy, said method comprising    administering, to a subject in need thereof, a pharmaceutically    active amount of at least one immunoglobulin single variable domain    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, VHH according to any of    aspects H-1 to H-22, polypeptide according to any of aspects K-1 to    K-19, compound or construct according to any of aspects L-1 to L-21,    monovalent construct according to any of aspects L-22 or L-23; or    composition according to aspect O-2 or O-3.-   Aspect R-5: Use of an immunoglobulin single variable domain    according to any of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1    to D-6, E-1 to E-13, F-1 to F-25 or G-1, a VHH according to any of    aspects H-1 to H-22, a polypeptide according to any of aspects K-1    to K-19, a compound or construct according to any of aspects L-1 to    L-21, or a monovalent construct according to any of aspects L-22 or    L-23 in the preparation of a pharmaceutical composition for    prevention and/or treatment of at least one variety of cancers;    and/or for use in one or more of the methods according to aspects    R-1 to R-3.-   Aspect R-6: Immunoglobulin single variable domain according to any    of aspects A-1 to A-22, B-1 to B-7, C-1 to C-4, D-1 to D-6, E-1 to    E-13, F-1 to F-25 or G-1, VHH according to any of aspects H-1 to    H-22, polypeptide according to any of aspects K-1 to K-19, compound    or construct according to any of aspects L-1 to L-21, monovalent    construct according to any of aspects L-22 or L-23; or composition    according to aspect O-2 or O-3 for the prevention and/or treatment    of at least one variety of cancers.

FIGURES

FIG. 1: nucleotide sequences encoding some of the amino acid sequencesand polypeptides of the invention.

FIG. 2: FACS binding data of HER3-specific Nanobodies and controlpolyclonal (PC) and monoclonal (MC) antibodies and an irrelevantNanobody (irr nb) to full length extracellular chicken HER3 or chimericchicken-human HER3.

FIGS. 3A to 3D show binding curves showing that the multivalent sequenceoptimized Nanobodies bind to HER3 but not to the other HER proteins (seeExample 25).

FIG. 4 gives the amino acid sequences of some of the proteins referredto in the present specification and claims.

FIGS. 5A and 5B show Western Blots showing the inhibition of pHER3 anddownstream signalling in BT-474 breast cancer cells by formattedsequence optimized Nanobodies (Example 28).

FIGS. 6A to 6C show Western Blots showing the inhibition of pHER3 anddownstream signalling in BxPC3 pancreatic cancer cells by formattedsequence optimized Nanobodies (Example 28).

FIGS. 7A to 7C show the inhibition of tumor growth (median tumor volumeover time) of A549 xenograft tumors treated with Nanobodies HER3MS00135(FIG. 7A), HER3MS00212 (FIG. 7B), and HER3MS00215 (FIG. 7C),respectively.

EXPERIMENTAL PART Example 1 Materials

1.1 hHER3 ECD (=Human HER3 Extracellular Domain)—SEQ ID NO: 4.

The gene encoding the human HER3 extracellular domain was generated byin-vitro synthesis. The open reading frame contained the coding sequenceof the cognate signal peptide of the HER3 gene followed by 624 aminoacids of the extracellular domain (ECD), and a C-terminal 6H is tag (seeSEQ ID NO: 4). The gene was cloned into the pEAK12d expression vector(Edge Biosystems). HER3ECD was produced by transient transfection ofHEK293-EBNA cells (Invitrogen). Briefly, cells adapted for suspensiongrowth in DMEM:F12 medium containing 4 ml/LInsulin-Transferrin-Selenium-X supplement and 1% Foetal Bovine Serum(all from Invitrogen) were incubated with a mixture of plasmid DNA andPoly-Ethylenelmine (PEI, PolySciences). After 90 min, transfected cellswere diluted 1:1 in Freestyle medium (Invitrogen) and placed on anorbital shaker at 37° C. in a 5% CO2 incubator under agitation at 160rpm. The supernatant was harvested after 6 days and sterile filteredthrough a 0.22 μm membrane cartridge (Millipore). The recombinantprotein was purified on a Poros 20 MC metal chelate affinitychromatography column (Applied Biosystems) charged with Ni ions,followed by size exclusion chromatography in PBS on a HiLoad Superdex 75prepgrade 16/60 column from GE Healthcare.

1.2 cHER3ECD (=Maccaca fascicularis HER3 Extracellular Domain=CynoHER3ECD)—SEQ ID NO: 3

The cyno HER3 sequence was determined by RT-PCR and cDNA sequencing. Theopen reading frame contained the coding sequence of the cognate signalpeptide of the cHER3 gene followed by 624 amino acids of theextracellular domain (ECD) and a C-terminal 6H is tag (SEQ ID No: 3).The gene was cloned into the pEAK12d expression vector (EdgeBiosystems).

The HER3ECD was produced by transient transfection of HEK293-EBNA cells(Invitrogen). Briefly, cells adapted for suspension growth in DMEM:F12medium containing 4 ml/L Insulin-Transferrin-Selenium-X supplement (allfrom Invitrogen) were incubated with plasmid DNA and Poly-Ethylenelmine(PEI, PolySciences) without pre-mixing. After 150 min, transfected cellswere diluted 1:3 in Freestyle medium (Invitrogen) and placed on anorbital shaker at 37° C. in a 5% CO2 incubator under agitation at 80 rpm(radius 2.5 cm). Temperature was lowered to 34° C. after 24 hincubation. The supernatant was harvested after 5 days and sterilefiltered through a 0.22 μm membrane cartridge (Millipore). Therecombinant protein was purified on HisTrap HP metal chelate affinitychromatography column (GE Healthcare) charged with Ni ions, followed bydialysis against PBS.

1.3 hHER3 Full Length Sequence—SEQ ID NO: 1

The human HER3 sequence (SEQ ID No: 1) was synthetically produced andcloned into pcDNA3.1 and pcDNA5/FRT (Invitrogen) respectively. The finalexpression plasmid pcDNA3.1-hHER3 was used to generate HER3-expressingtransfected cell lines.

1.4 Generation HER3 Expressing Transfectants

HEK 293T cells (DSMZ) were transient transfected with pcDNA3.1-hHER3using Fugene HD (Roche) as transfection agent. Transfected cells wereused to immunize llamas. Chinese hamster ovary cells (ATCC) weretransfected with pcDNA3.1-hHER3, single cell sorted and selected forhigh and homogenous expression of HER3 by staining the cells using aHER3-specific monoclonal antibody (R&D Systems) and FACS analysis. Oneclone was selected and transfected with pcDNA3.1-hygro encoding humanHER2 (SEQ ID NO: 9) to obtain HER2/HER3 double transfected cells. Thecells were single cell sorted and a clone was selected with high andhomogenous expression of HER2 by staining the cells using aHER2-specific monoclonal antibody (R&D Systems) and FACS analysis. Thesecells were used in binding and competition experiments.

FlpIn CHO cells (Invitrogen, #R-758-7) were transfected withpcDNA5/FRT-hHER3 plasmid and grown under hygromycin selection.

A camel cell line (CAKI; Nguyen et al. 2001. Adv. Immunol. 79: 261-296)was transfected with pcDNA3.1-hHER3 (SEQ ID NO: 1) and a single cellsorted clone was selected and used in selection experiments.

Example 2 Identification of HER3-Specific Nanobodies

2.1 Immunizations

After approval of the Ethical Committee of the Faculty of VeterinaryMedicine (University Ghent, Belgium), 8 llamas were immunized, accordingto standard protocols. Three llamas (340, 342, 345) received 4intramuscular injections at biweekly intervals of in-house made HER3-ECD(ECD: extracellular domain) SEQ ID NO: 4 with a dose of 100, 50, 25 and25 microgram. Five llamas (419, 420, 421, 429, 430) received 4subcutaneous injections at biweekly intervals of 2×10⁷ transienttransfected HEK293-HER3 cells.

2.2 Evaluation of Induced Responses in Llama

At the end of the immunization procedure, sera samples were collectedfrom all animals to evaluate the induction of immune responses againstHER3 by ELISA. In short, recombinant human HER3/Fc chimera wasimmobilized in a 96 well Maxisorp plate (Nunc, Wiesbaden, Germany).After blocking and addition of serum dilutions, specifically boundimmunoglobulins were detected using monoclonal antibodies specific tollama IgG1, IgG2 or IgG3 and rabbit anti-mouse horseradish peroxidaseconjugate. A significant HER3 specific immune response was observed inall animals. The antibody response was mounted both by the conventionaland heavy chain only antibody B-cell repertoires since specificallybound immunoglobulins could be detected with antibodies specificallyrecognizing the conventional llama IgG1 antibodies or the heavy-chainonly llama IgG2 and IgG3 antibodies.

TABLE C-1 Overview of the HER3-specific serum titers. The serum titer isdefined as the highest serum dilution that results in a signal to noise1 ≥ 2 Llama ID IgG1 IgG2 IgG3 340 - day 50 1.09E+06 3.65E+05 3.65E+05342 - day 50 1.09E+06 1.22E+05 3.65E+05 345 - day 50 1.09E+06 4.05E+044.05E+04 419 - day 64 4.05E+04 1.50E+03 5.00E+02 420 - day 64 1.50E+03<500 <500 421 - day 64 1.35E+04 <500 <500 429 - day 49 1.35E+04 <500<500 430 - day 49 4.05E+04 <500 <500 ¹Signal to noise is defined as theratio of OD450 nm absorptions of day blood collection after immunization(day 49, 50 or 64) versus pre-immune (day 0) serum sample.2.3 Library Construction

Peripheral blood mononuclear cells were prepared from the blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. TotalRNA extracted from these cells and from lymph nodes was used as startingmaterial for RT-PCR to amplify Nanobody encoding gene fragments. Thesefragments were cloned into phagemid vector pAX50. Phage was preparedaccording to standard protocols (Phage Display of Peptides and Proteins:A Laboratory Manual, Academic Press; 1st edition (Oct. 28, 1996)) andstored after filter sterilization at 4° C. until further use. In total,8 phage libraries were constructed (340, 342, 345, 419, 420, 421, 429and 430), with library sizes between 3×10⁸ and 8.5×10⁸, and a percentageof insert ranging from 91 to 100%.

2.4 Selections in Search of HER3-Specific Nanobodies

To identify Nanobodies recognizing human HER3, the phage libraries wereincubated with soluble biotinylated HER3-ECD (SEQ ID NO: 4). The proteinwas produced as described in Example 1 and biotinylated usingSulfo-NHS-LC-Biotin (Pierce). Complexes of biotinylated HER3 and phagewere captured from solution on streptavidin coated magnetic beads. Afterextensive washing with PBS/0.05% Tween20, bound phage were eluted byaddition of 1 mg/ml trypsin or 1 μM HRG (R&D systems). The phagelibraries 340, 342 and 345 were incubated with soluble biotinylatedhuman HER3-ECD (0.1-100 nM); phage libraries 419, 420, 421, 429 and 430with soluble biotinylated human HER3-ECD (1-10-100-1000 nM) in twoconsecutive rounds. Outputs of these selections were analyzed forenrichment factor (number of phage present in eluate relative tocontrols) and individual clones from these first round outputs werepicked. All phage libraries were also incubated with a Chinese hamsterovary (ATCC) or camel cell line (CAKI cells; Nguyen et al. 2001. Adv.Immunol. 79: 261-296)) transfected with hHER3 (hHER3=human HER3=SEQ IDNO: 1) or with hHER2 and hHER3 (hHER2=human HER2=SEQ ID NO: 9) (5×10⁶cells) in two consecutive rounds. A third selection strategy consistedof coating plates with a HER3-specific Nanobody (04C07 and 21F06; 10μg/ml), capture of HER3-ECD (5-100 nM) and addition of the phagelibraries in order to enrich phages binding to different epitopes.Trypsin was used to elute phages and outputs were used as input for asecond round selection on plates coated with the same or the alternativeNanobody. Individual clones were picked from the different selectionconditions.

All individual clones were grown in 96 deep well plates (1 ml volume).Nanobody expression was induced by adding IPTG to a final concentrationof 1 mM. Periplasmic extracts were prepared by freezing the cell pelletsand dissolving them in 100 μl PBS. Cell debris was removed bycentrifugation.

2.5 Screening for Nanobodies Binding hHER3

To determine the binding capacity of Nanobodies to hHER3, periplasmicextracts were screened in a cell based binding FACS assay (FACS Array,BectonDickinson). Samples were incubated with Chinese hamster ovarycells transfected with hHER3 or hHER2/hHER3, washed with FACS buffer andbinding of the Nanobodies was detected using an anti-c-myc-specificantibody (Serotec). Nanobodies binding to hHER3 were identified.

2.6 Screening for Nanobodies Competing with HRG1-β1 Binding

To determine the HRG1-β1 (hHRG=human Heregulin) blocking capacity of theNanobodies, periplasmic extracts were screened in a cell basedcompetition assay using the FMAT technology (Applied Biosystems, FosterCity, Calif.). HRG1-β1-EGF (R&D Systems, #396-HB, Accession # NP_039250)was labelled with A647 and incubated with Chinese hamster ovary cellstransfected with hHER2/hHER3 in the presence of Nanobodies. Decrease intotal FL1 signals indicates that the binding of labelled HRG1-β1 isblocked by the Nanobody present in the periplasmic extract. Nanobodieswere identified with different levels of blocking the ligand-receptorinteraction, ranging from 100% block to no block. Based on HER3 bindingand HRG1-β1 competition screening, a set of HER3 Nanobodies was selectedand sequenced. Sequence analysis revealed 204 different families of HER3specific Nanobodies.

2.7 Surface Plasmon Resonance Analysis of Periplasmic Extracts on hHER3

Off-rates of the periplasmic extracts containing anti-HER3 Nanobodieswere measured by Surface Plasmon Resonance (SPR) using a Biacore T100instrument. Human HER3-ECD (SEQ ID NO: 4) was covalently bound to a CMsensor chip surface via amine coupling using EDC/NHS for activation andethanolamine HCl for deactivation. Periplasmic extracts containingHER3-specific Nanobodies were injected for 2 minutes at a flow rate of45 μl/min to allow binding to chip-bound antigen. Next, binding bufferwithout periplasmic extracts was sent over the chip at the same flowrate to allow spontaneous dissociation of bound Nanobody. From thesensorgrams obtained for the different periplasmic extractsk_(off)-values (k_(d)) were calculated.

2.8 Screening for Nanobodies Inhibiting Ligand Dependent HER3Phosphorylation

To identify HER3-specific Nanobodies with a capacity to block HER3phosphorylation, periplasmic extracts were incubated with serum-starvedMCF-7 cells followed by 5 nM HRG1-β1-EGF stimulation for 15 minutes.Cell lysates were made and phosphorylation of HER3 was measured usingthe DuoSet IC human phospho-HER3ELISA (R&D systems, DYC1769-2).Nanobodies were identified with different levels of inhibiting theligand-induced pHER3, ranging from 100% block to no inhibition.

2.9 Epitope Binning of Nanobodies

Six HER3-specific Nanobodies were biotinylated and used in alphascreenand/or FACS competition assays to group Nanobody families in differentepitope bins. In the FACS assay, periplasmic extracts containingHER3-specific Nanobodies were incubated in presence of one out of thesix biotinylated Nanobodies (used concentrations are indicated in TableC-2). The incubation mixtures were subsequently added to HER2/HER3transfected CHO cells. After 90 minutes incubation, the cells werewashed and binding of the biotinylated Nanobody was detected usingstreptavidin-PE. The obtained fluorescent signal is compared to thesignal obtained from a condition where the biotinylated Nanobody wasadded to the cells without periplasmic extract Nanobody.

For the alphascreen competition assay, human HER3-Fc (R&D Systems,348-RB) was captured on anti-human Fc Nanobody conjugated Acceptor beadswhich were prepared according to the manufacturer's instructions(PerkinElmer). To evaluate the blocking capacity of anti-HER3Nanobodies, dilutions of the periplasmic extracts were added to one ofthe biotinylated Nanobodies (see Table C-2). To this mixture, humanHER3-Fc-Acceptor beads were added and incubated for 1 hour at roomtemperature. Then, the streptavidin-coupled Donor beads were added andfurther incubated for 1 hour at room temperature. Fluorescence wasmeasured using the EnVision Multilabel Plate Reader (PerkinElmer) usingan excitation wavelength of 680 nm and an emission wavelength of 520 nm.

TABLE C-2 Concentrations of biotinylated Nanobodies used in epitopecompetition FACS and alphascreen and concentrations human HER3-Fc usedin alphascreen. FACS Alphascreen Concentration Concentrationbiotinylated Concentration biotinylated Nanobody hHER3-Fc Nanobody 04C073 nM 0.26 nM  0.1 nM 21B02 0.9 nM 0.26 nM 0.26 nM 23F05 0.83 nM  0.1 nM0.04 nM 18E08 60 nM  1.6 nM   4 nM 17C08 20 nM NA NA 04F10 100 nM NA NANA: not applicable

Non-competing Nanobodies were grouped in different epitope groups. Fivedifferent groups were identified based on the alphascreen and FACScompetition results.

Example 3 Expression and Purification of hHER3-Specific Nanobodies

Based on the described screening assays, 15 Nanobodies were selected forfurther characterization. These Nanobodies belong to 13 differentfamilies and five different epitope bins. Sequences are shown in TableA-1 (SEQ ID NOs: 12 to 26).

Nanobodies were expressed in E. coli TG1 cells as c-myc, His6-taggedproteins in a culture volume of 500 mL. Expression was induced byaddition of 1 mM IPTG and allowed to continue for 3 h at 37° C. Afterspinning the cell cultures, periplasmic extracts were prepared byfreeze-thawing the pellets and resuspension in dPBS. These extracts wereused as starting material for immobilized metal affinity chromatography(IMAC) using Histrap FF crude columns (GE Healthcare). Nanobodies wereeluted from the column with 250 mM imidazole and subsequently desaltedtowards dPBS (Dulbecco's Phosphate Buffered Saline).

Example 4 Binding Capacity of Purified Nanobodies to hHER3 in ELISA

The binding capacity of 14 purified Nanobodies belonging to 5 differentepitope bins was determined in ELISA. 96-well plates were coated withhHER3-ECD (1 μg/ml). A dilution series of each Nanobody starting from500 nM down to 6 pM was tested and detected using mouse anti c-myc(Roche) and anti-mouse-HRP (Dako cytomation). All Nanobodies bind tohHER3-ECD and the obtained EC50 values are shown in Table C-3.

TABLE C-3 EC50 values for various anti-HER3 Nanobodies to hHER3-ECD andtheir 95% confidence intervals (CI) as determined by ELISA SEQ ID NOEC50 (nM) CI95 04C07 15 0.86 0.5-1.6 05A09 19 1.45 0.92-2.3  17B05 130.63 0.35-1.13 17C08 20 0.62  0.4-0.96 17E08 25 1.02 0.48-2.15 18B05 140.4  0.2-0.83 18E08 17 4.7 3.3-6.8 18F05 12 0.29 0.15-0.55 18G11 16 0.720.39-1.33 21B02 21 1.1 0.7-1.6 21F06 22 0.3 0.14-0.65 23F05 23 0.460.28-0.77 34A04 24 0.57 0.35-0.92 34C07 18 0.46 0.26-0.8 

Example 5 Affinity of Purified Nanobodies

Affinity measurements were performed using a Biacore T100 instrument bycoating anti-human Fc antibody (GE Healthcare) to a CM sensorchipsurface via amine coupling using EDC/NHS for activation and ethanolamineHCl for deactivation. HER3-Fc (R&D systems, 348-RB 5 μg/ml; 120 s; 10μl/min) was injected to allow capturing by the coated anti-Fc antibody.Then, purified Nanobodies were injected for 2 minutes at a flow rate of10 μl/min to allow binding to chip-bound antigen. Next, binding bufferwithout Nanobodies was sent over the chip at the same flow rate to allowspontaneous dissociation of bound Nanobody. The kinetic parametersk_(on)-values (k_(a)), k_(off)-values (k_(d)) and K_(D) were calculatedfrom the sensorgrams obtained for the different Nanobodies (Table C-4).

TABLE C-4 Kinetic parameters for purified HER3-specific Nanobodies.k_(a) (1/Ms) k_(d) (1/Ms) KD (nM) 04C07 1.20E+06 2.60E−04 0.22 17B053.90E+05 4.30E−05 0.11 17C08* 8.40E+03 4.10E−04 48 17E08 3.63E+054.10E−05 0.11 18B05 4.90E+05 1.42E−04 0.29 18E08 1.04E+04 5.61E−04 53.818F05 4.27E+05 1.04E−04 0.24 18G11 2.60E+04 2.70E−04 10 21B02 2.20E+062.60E−03 1.22 21F06 1.80E+07 2.20E−03 0.12 23F05 3.10E+06 3.10E−03 0.9934A04 2.63E+06 7.06E−03 2.7 34C07** 1.80E+04 2.50E−04 14 Control Fab17.03E+05 3.98E−04 0.57 Control Fab2 9.34E+04 2.59E−04 2.77 *Heterogenouscurve: results of main interaction (89%) presented **Heterogenous curve:results of main interaction (84%) presented

Example 6 Binding Capacity of Purified Nanobodies to cynoHER3 in ELISA

The binding capacity of the selected purified Nanobodies was determinedin ELISA. 96-well plates were coated with cHER3-ECD (1 μg/ml) (SEQ IDNO: 4). A dilution series of each Nanobody starting from 500 nM down to6 pM was tested and detected using mouse anti c-myc (Roche) andanti-mouse-HRP (Dako cytomation). All Nanobodies bind to cyno HER3-ECDand the obtained EC50 values are shown in Table C-5.

TABLE C-5 EC50 values for various anti-HER3 Nanobodies to cyno HER3- ECDand their 95% confidence intervals as determined by ELISA EC50 (nM) CI9504C07 0.93 0.5-1.6 05A09 1.3 0.83-2.03 17B05 0.66  0.4-1.08 17C08 0.590.39-0.89 17E08 1.15 0.65-2.05 18B05 0.45 0.26-0.78 18E08 4.4 3.4-5.718F05 0.29 0.16-0.55 18G11 0.64 0.36-1.13 21B02 0.9 0.6-1.3 21F06 0.320.15-0.68 23F05 0.44 0.26-0.74 34A04 0.49 0.31-0.78 34C07 0.43 0.24-0.76

Example 7 HER3-Specificity of Purified Nanobodies

Off-target binding of purified HER3 Nanobodies was assessed by measuringtheir binding capacity to Chinese hamster ovary (CHO) cells transfectedwith human HER1 (SEQ ID NO: 8), human HER2 (SEQ ID NO: 9) or human HER4(SEQ ID NO: 10) by FACS. Non-transfected cells were used to checkbinding to the cell background. Purified HER3-specific Nanobodies(2000-666-222 nM) were added to 2×10⁵ cells, 30 minutes incubated at 4°C. and detected using mouse anti-c-myc (Serotec) and goat anti-mouse-PE(Jackson Immuno-Research Laboratories). Binding of polyclonal antibodies(anti-HER1, R&D Systems AF231; anti-HER2, R&D Systems AF1129; anti-HER4,R&D Systems AF1131) were used as positive control. Low binding levelswere observed for Nanobody 21B02 to HER2, whereas the other purifiedNanobodies did not bind to HER1, HER2 and HER4 transfected cells (TableC-6).

TABLE C-6 Binding of purified Nanobodies and control polyclonalantibodies to Chinese hamster ovary ovary cells transfected with HER1,HER2 or HER4 as determined by FACS (data represent MCF values). HER1HER2 HER4 2000 nM 667 nM 222 nM 2000 nM 667 nM 222 nM 2000 nM 667 nM 222nM Nb Nb Nb Nb Nb Nb Nb Nb Nb 04C07 72 92 75 154 79 84 105 103 96 17B0549 64 85 134 86 84 116 90 142 17C08 58 47 62 1218 418 160 254 91 21017E08 59 52 66 73 51 51 242 117 110 18B05 61 101 82 77 63 61 96 95 6018E08 64 62 61 78 52 53 115 80 91 18F05 133 60 154 256 347 436 120 149149 18G11 97 87 101 65 117 109 124 56 57 19E03 91 104 81 167 78 99 140110 77 21B02 53 64 64 8574 3609 1342 273 119 91 21F06 74 110 107 409 22984 263 225 149 23F05 67 67 153 832 264 97 95 101 343 Anti-HER1 pAb (5μg/ml) 28217 888 418 Anti-HER2 pAb (5 μg/ml) 323 41048 578 Anti-HER4 pAb(5 μg/ml) 584 932 16981

Example 8 Epitope Mapping of Purified Nanobody Panel

The classification of Nanobodies in the different epitope bins was animportant criterium to select Nanobodies for purification and furthercharacterization (see Examples 2.9 and 3). The FACS competition assayusing the biotinylated Nanobodies 04C07 (also denoted as 4C07), 21B02,23F05, 18E08, 17C08 and 04F10 (also denoted as 4F10) was repeated withthree different concentrations of 14 selected purified Nanobodies (400,100 and 25 nM). The Nanobodies belonging to group 1 and group 2 are verysimilar but differ in their capacity to compete with Nanobody 04F10(Table C-7). The Nanobodies of group 3 and group 4 are also very similarbut differ in their capacity to compete with 04F10 and 17C08. TheNanobodies classified in one epitope group are considered to bind toidentical epitopes, whereas Nanobodies of different epitope groups areconsidered to bind to partially overlapping (group 1-2 and group 3-4) ornon-overlapping epitopes.

TABLE C-7 Epitope competition FACS of 14 selected purified Nanobodiesagainst biotinylated Nanobodies. The data represent the percentagecompetition at a 400 nM concentration of the non-biotinylated Nanobody.Epi- Nano- 04C07- 21B02- 23F05- 18E08- 17C08- 04F10- tope body biotinbiotin biotin biotin biotin biotin group 17B05 100 0 0 0 0 68 1 17E08100 5 0 0 0 32 1 18B05 98 0 0 0 0 31 1 18F05 100 0 0 0 0 15 1 04C07 1002 0 26 15 0 2 05A09 0 22 10 78 48 0 3 18E08 0 4 0 86 63 0 3 18G11 0 0 097 74 0 3 34C07 0 6 10 99 56 0 3 17C08 3 16 17 98 97 77 4 21B02 10 10098 20 17 15 5 21F06 11 100 100 ND 9 15 5 23F05 6 100 100 16 8 15 5 34A0422 99 97 25 14 3 5 ND: not determined

To obtain more insight in the epitopes recognized by selected purifiedNanobodies, various human-chicken HER3 chimeric proteins wereengineered, based on the division of the HER3 extracellular domain intofour distinct domains. Plasmids were constructed which encode thetransmembrane and extracellular regions of chicken HER3 (based onsequence Genbank accession nr: DQ358720) and variants with individualdomains from human HER3 swapped into the chicken scaffold but using thehuman signal sequence:

-   -   Chicken Her3: AA1-25 human, AA26-642 chicken (SEQ ID NO: 2)    -   Chimeric chicken Her3-human domain 1: AA1-206 human, AA207-642        chicken (SEQ ID NO: 5)    -   Chimeric chicken Her3-human domain 2: AA1-25 human, AA26-206        chicken, AA207-328 human, AA329-642 chicken (SEQ ID NO: 6)    -   Chimeric chicken Her3-human domain 4: AA1-25 human, AA26-495        chicken, AA496-642 human (SEQ ID NO: 7)

The HER3-encoding fragments were synthetically produced and cloned inpcDNA3.1 (Invitrogen). HEK293 (DSMZ) cells were transient transfectedwith chicken or chimeric HER3 constructs. Surface expression of theconstructs was confirmed using a goat anti-hHER3 polyclonal antibody(R&D systems). The Nanobodies were incubated with the transienttransfected cells and binding was detected using anti-c-myc and goatanti-mouse-PE. Result of the binding study is shown in FIG. 2. Insummary, Nanobodies 17B05, 17E08, 18B05, 18F05 and 04C07 (epitope groups1 and 2) recognize human domain 1 while Nanobodies 18E08 and 18G11(epitope group 3) bind to human domain 2. Nanobody 17C08 (epitope group4) recognizes both domain 1 and domain 2. Three Nanobodies (21B02, 21F06and 23F05 (epitope group 5)) showed chicken cross-reactivity, hencecorresponding epitopes could not be mapped to a specific human HER3domain.

Domain 1 is involved in ligand binding whereas the dimerization loopinvolved in HER dimerization is located in domain 2 (Baselga and Swain,2009 Nature Reviews Cancer Vol 9, p 463-475). Therefore, the mode ofaction of Nanobodies binding to domain 1 could be related withinhibition of ligand binding and that of Nanobodies binding to domain 2could be related with blocking HER dimerization

Example 9 HRG1-β1 Competition Capacity of Purified Nanobodies in FACS

The HRG1-β1 competition capacity of purified HER3-specific Nanobodieswas determined in a FACS competition experiment using Chinese hamsterovary cells (CHO FlpIn, Invitrogen) transfected with hHER3. A dilutionseries of each Nanobody starting from 600 nM down to 0.03 nM and 0.8 nMHRG1-β1-EGF (R&D systems, #396-HB) were added to the cells (2×10⁵) andincubated during 90 minutes at 4° C. After washing the cells with FACSbuffer, detection was performed using goat anti-HRG ECD (R&D systems)and donkey anti-goat PE (Jackson ImmunoResearch Laboratories).Nanobodies showed complete competition with HRG1-β1-EGF for HER3binding, except for Nanobodies 18E08 and 18G11 which did not compete andNanobody 17C08 which only partially blocked (75%) at a Nanobodyconcentration of 600 nM. The obtained IC50 values are shown in TableC-8. The lower HRG1-β1 blocking capacity of Nanobodies belonging toepitope group 3 and in lesser extent also group 4 is in agreement withtheir binding to domain 2 and blocking HER transphosphorylation as modeof action.

TABLE C-8 IC50 values for competition between HRG1-β1-EGF and variousanti-HER3 Nanobodies to CHO cells transfected with HER3 and their 95%confidence intervals as determined by FACS. IC50 (nM) CI95 04C07 4.263.13-5.81 17B05 33.4 29.04-38.40 17C08 Partial competition NA 17E08 75.964.94-88.73 18B05 16.15 11.80-22.09 18E08 No competition NA 18F05 20.1115.63-25.88 18G11 No competition NA 21B02 13.29  9.09-19.44 21F06 10.1 7.44-13.71 23F05 9.44  8.20-10.88 Control MAb1 9.4  7.5-11.8 ControlMAb2 67.2 46.4-97.3 NA: not applicable.

Example 10 Inhibition of HER3, Akt and ERK1/2 Phosphorylation bypurified Nanobodies

To determine the potency of monovalent Nanobodies in inhibiting ligandinduced HER3 phosphorylation and downstream signaling, Nanobodies weretested in cell based assays as described below.

Inhibition of ligand induced pHER3 (phosphoHER3) in MCF-7 cells (cellbased electrochemiluminescence assay (ECLA)): MCF-7 cells (ATCC HTB 22)were serum starved and pre-incubated with Nanobodies (serial dilutions,starting concentration 666 nM) in serum-free media for 60 min at 37° C.,5% CO₂. Cells were stimulated with 50 ng/ml HRG1-β1 EGF domain (R&DSystems, #396-HB) for 10 min, supernatants discarded and cells lysed incold NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH8.0, 137 mM NaCl, 10%glycerol, 2 mM EDTA, protease inhibitor cocktail set III (Calbiochem),phosphatase inhibitor cocktail set II (Calbiochem)). MA6000 96 wellplates (MSD, # L15XB) were blocked with 3% block A (MSD) in PBS, pH7.4,0.05% Tween20 and coated with HER3 specific capture antibody (R&DSystems, # MAB3481). Cell lysates were added and incubated for 2 h atroom temperature (RT). Biotinylated anti-phospho Tyrosine antibody (R&DSystems, # BAM1676) and sulfo tag streptavidin reagent (MSD, #R32AD)were used for detection (Table C-9).

10.1 Inhibition of Downstream Signaling (pAkt/pERK1/2):

MCF-7 cells were pre-incubated with Nanobodies and stimulated asdescribed above. Cell lysates were tested in Phospho-Akt (Ser473) WholeCell Lysate Kit (MSD, # K151CAD) and Phospho-ERK1/2 Whole Cell LysateKit (MSD, # K111DWD) according to manufacturer's instructions (TableC-10).

10.2 Inhibition of EGFR/HER3 and HER2/HER3 Transphosphorylation: MDAMB468 (ATCC HTB 132) and CHO HER2/HER3 cells were serum starved andtreated as described above (MDA MB468 stimulation with 50 ng/ml HRG1-β1EGF domain; CHO HER2/HER3 cells stimulation with 100 ng/ml HRG1-β1 EGFdomain). Cell lysates were tested in pHER3 ECLA (Table C-9).

TABLE C-9 Inhibition of HRG1-β1 dependent HER3 phosphorylation bypurified Nanobodies CHO-HER2/HER3 MDA-MB468 Nanobody MCF-7 (IC50 M)(IC50 M) (IC50 M) 04C07 1.07E−09 2.407E−09  6.68E−09 17B05 5.34E−091.18E−09 2.79E−08 18B05 5.18E−09 3.87E−09 3.00E−08 18F05 1.53E−093.03E−09 1.80E−08 17E08 4.51E−09 8.01E−10 2.46E−08 18E08 6.39E−08 <50%1.01E−07 18G11 1.03E−08 2.13E−08 7.03E−09 05A09 9.12E−08 <50% 1.28E−0734C07 1.89E−08 3.53E−08 1.07E−08 21B02 4.88E−08 <50% 9.99E−08 21F066.789E−09  1.447E−08  7.94E−09 23F05 1.77E−08 7.63E−08 2.41E−08 34A043.02E−08 1.34E−07 2.205E−08  17C08 8.98E−08 <50% 5.38E−08 <50% = lessthan 50% inhibition

TABLE C-10 Inhibition of HRG1-β1 induced pAKT and pERK1/2 Nanobody pAkt(IC₅₀ M) pERK1/2 (IC₅₀ M) 04C07 1.09E−08 2.79E−08 17B05 7.83E−092.36E−09 18B05 6.02E−09 <50% 18F05 8.04E−09 8.98E−08 17E08 5.78E−092.78E−09 18E08 5.69E−08 <50% 18G11 1.38E−07 <50% 05A09 2.898E−07  <50%34C07 4.99E−08 1.00E−07 21B02 6.90E−08 <50% 21F06 2.31E−08 1.11E−0723F05 4.648E−08  <50% 34A04 6.255E−08  3.13E−07 17C08 7.24E−07 <50% <50%= less than 50% inhibition

Purified Nanobodies potently inhibited HRG1-131 induced HER3phosphorylation in MCF-7 cells, and cells expressing EGFR/HER3 (MDAMB468) or HER2/HER3 respectively (CHO HER2/HER3) with the exceptions of18E08, 05A09, 21B02, and 17C08 which showed less than 50% inhibition ofpHER3 in CHO HER2/HER3 cells. PI3Kinase pathway (pAkt) inhibition couldbe detected with all Nanobodies. To a lesser extent downstream signalingthrough pERK1/2 could be blocked by Nanobodies.

The data described in Examples 9 and 10 suggest that Nanobodies of group3 block transphosphorylation of HER3 without blocking ligand binding.

Example 11 Migration Blocking Capacity of Purified Nanobodies

The ability of Nanobodies to inhibit HRG1-β1 dependent cell migrationwas assessed in the following assay. A431 cells (CRL 1555) were seededin HTS Fluoroblok 96 well plate inserts (BD Falcon #351164) in thepresence of Nanobodies (serial dilutions; starting at 666 nM). Mediaplus 500 nM HRG1-β1 extracellular domain (R&D Systems, #377-HB) wasadded to the bottom wells. Migrated cells were stained with CalceinAMand fluorescence detected by plate reader. Nanobodies inhibiting liganddependent cancer cell migration could be identified (Table C-11).

TABLE C-11 Inhibition of HRG1-β1 induced cell migration Nanobody IC₅₀ M04C07 6.52E−09 17B05 3.04E−09 18B05 <50% 18F05 1.52E−07 17E08 5.76E−0818E08 <50% 21B02 <50% 21F06 4.00E−08 23F05 3.68E−07 04F10 no effect17C08 no effect 18G11 6.79E−08 34C07 1.37E−07 <50% = less than 50%inhibition

Example 12 Generation of Formatted Nanobodies

The structural requirement for multi-specificity is to fuse two or morebinding domains together, with sufficient flexibility to allowsimultaneous binding to different target epitopes and/or combine bindingdomains with a different mode of action (blocking ligand binding andblocking HER dimerization) in one molecule.

Nanobodies binding to different epitopes were combined in one moleculeand fused to an anti-albumin binding Nanobody to increase the half-lifeof the molecule. GS-linkers were inserted between the Nanobody buildingblocks. Binding of the two HER3-specific Nanobody building blockssimultaneously without a significant loss of entropy increased bindingaffinity to the target, resulting in higher potency and/or higherspecificity. Careful selection of the epitopes targeted on the antigenand optimal design of linkers to allow maximal flexibility of thebinding domains resulted in the blocking of two or more critical sitesof the target. The amino acid sequences of selected formatted Nanobodyconstructs are shown in Table A-2.

Example 13 Analysis of the HER1-HER3 Heterodimerization BlockingCapacity of Monovalent Nanobody 17B05

To determine the potency of monovalent Nanobodies in inhibiting EGFRligand induced HER3 phosphorylation Nanobodies were tested in cell basedassays as described below.

Inhibition of EGFR ligand induced pHER3 (phosphoHER3) in CHO EGFR/Her3cells (cell based electrochemiluminescence assay (ECLA)): CHO EGFR/Her3were serum starved and pre-incubated with Nanobodies or EGFR antibodycetuximab (serial dilutions, starting concentration 666 nM or 167 nM) inserum-free media for 60 min at 37° C., 5% CO₂. Cells were stimulatedwith 100 ng/ml rhTGFα (R&D Systems, #239-A), EGF (Sigma#E-9644), or rhepiregulin (R&D Systems, #1195EP) respectively for 10 min, supernatantsdiscarded and cells lysed in cold NP-40 lysis buffer (1% NP-40, 20 mMTris, pH8.0, 137 mM NaCl, 10% glycerole, 2 mM EDTA, protease inhibitorcocktail set III (Calbiochem), phosphatase inhibitor cocktail set II(Calbiochem)). MA6000 96 well plates (MSD, # L15XB) were blocked with 3%block A (MSD) in PBS, pH7.4, 0.05% Tween20 and coated with HER3 specificcapture antibody (R&D Systems, # MAB3481). Cell lysates were added andincubated for 2 h at room temperature (RT). Biotinylated anti-phosphoTyrosine antibody (R&D Systems, # BAM1676) and sulfo tag streptavidinreagent (MSD, #R32AD) were used for detection (Table C-12). MonovalentNanobody 17B05 was able to potently block EGFR ligand induced HER3phosphorylation, whereas domain II binders 18G11 and 34C07 were not ableto block EGFR ligand induced HER3 phosphorylation.

TABLE C-12 Inhibition of EGFR ligand induced HER3 phosphorylation CHOCHO CHO EGFR/HER3 + EGFR/HER3 + EGFR/HER3 + TGFα EGF epiregulinNanobody/antibody (IC50 M) (IC50 M) (IC50 M) cetuximab 1.09e−0091.29e−009 6.35e−010 18G11 no effect no effect no effect 34C07 no effectno effect no effect 17B05 8.15e−010 3.43e−010 2.48e−010 control Mab-1 noeffect no effect <50% control Mab-2 no effect no effect 2.36e−007 <50% =less than 50% inhibition

Example 14 Generation of Multivalent Parental HER3-Specific Nanobodieswith Half-Life Extension (HLE)

In order to generate a half-life extended Nanobody product that blocksHRG binding to HER3 and also blocks heterodimerization of HER3, themonovalent lead panel Nanobodies (4C07, 17C08, 18G11, 21F06, 34C07 and17B05) were formatted to bivalent and multivalent molecules.

For half-life extension, it was opted to fuse the constructs to theanti-HSA Nanobody ALB11.A library approach was used to make all possibleNanobody1-35GS-Nanobody2-9GS-ALB11 combinations. The multivalentNanobodies were expressed as c-myc, His6-tagged protein in Pichiapastoris. Induction of Nanobody expression occurred by stepwise additionof methanol. Clarified medium with secreted Nanobody was used asstarting material for immobilized metal affinity chromatography (IMAC)followed by desalting resulting in at least 90% purity as assessed bySDS-PAGE. The purified multivalent parental HER3-specific Nanobodieswere tested in a HRG-blocking alphascreen using 20 nM HRG and 0.05 nMHER3-Fc. The results indicated that the best Nanobody building blockdetermined the potency of the formatted Nanobodies (Table C-13).Nanobody 21F06 was the most potent monovalent building block in thisassay (IC50 1.35E-11 M) and all constructs containing 21F06 as buildingblock have IC50s between 5.69E-12 M and 5.77E-11 M.

TABLE C-13 HRG competition alphascreen results of multivalent parentalanti-HER3 Nanobodies construct Nb1 linker Nb2 linker ALB11 IC50 (M) CI95(M) % inhibition HER3MS00022 21F06 35GS 21F06 9GS ALB11 5.69E−12  [4E−12, 8E−12]   103% HER3MS00024 21F06 35GS 4C07 9GS ALB11 8.98E−12  [6E−12, 13E−12]   104% HER3MS00034 21F06 35GS 18G11 9GS ALB11 1.06E−11 [0.8E−11, 1.3E−11] 99.40% HER3MS21F06 21F06 1.35E−11  [0.5E−11,2.9E−11] 103.7% HER3MS00060 21F06 35GS 17B05 9GS ALB11  1.2E−11 [9.4E−12, 1.4E−11]   108% HER3MS00023 21F06 35GS 34C07 9GS ALB111.57E−11  [1.3E−11, 1.9E−11]   102% HER3MS00030 4C07 35GS 21F06 9GSALB11 1.58E−11  [1.4E−11, 1.8E−11] 101.70%  HER3MS00037 18G11 35GS 18G119GS ALB11 1.59E−11  [1.2E−11, 2.1E−11]   107% HER3MS00061 17B05 35GS21F06 9GS ALB11  2.5E−11  [1.9E−11, 3.1-11]   108% HER3MS00026 34C0735GS 21F06 9GS ALB11 5.77E−11  [4.4E−11, 7.6E−11]   104% HER3MS000554C07 35GS 17B05 9GS ALB11  1.2E−10  [8.9E−11, 1.5E−10]   87% HER3MS000324C07 35GS 4C07 9GS ALB11 1.32E−10 [1.14E−10, 1.52E−10] 86.60%HER3MS00035 4C07 35GS 18G11 9GS ALB11 1.34E−10 [1.08E−11, 1.67E−11]90.40% HER3MS00031 4C07 35GS 34C07 9GS ALB11 1.57E−10 [1.15E−10,2.14E−10] 87.40% HER3MS00039 18G11 35GS 4C07 9GS ALB11 2.14E−10[1.72E−10, 2.68E−10] 90.70% HER3MS00057 17B05 35GS 4C07 9GS ALB11 2.2E−10  [1.7E−10, 2.9E−10]   88% HER3MS04C07 4C07 2.64E−10  [2.6E−10,3.2E−10] 86.70% HER3MS00056 17B05 35GS 17B05 9GS ALB11  3.1E−10 [2.3E−10, 4.2E−10]   88% HER3MS00054 17B05 35GS 34C07 9GS ALB11 3.2E−10  [2.8E−10, 3.6E−10]   89% HER3MS00028 34C07 35GS 4C07 9GS ALB113.27E−10  [2.4E−10, 4.5E−10] 88.60% HER3MS00058 17B05 35GS 18G11 9GSALB11  4.2E−10  [3.4E−10, 5.2E−10]   96% HER3MS00051 34C07 35GS 17B059GS ALB11  4.6E−10  [3.6E−10, 5.7E−10]   95% HER3MS00038 18G11 35GS21F06 9GS ALB11 4.76E−10 [3.51E−10, 6.46E−10]   96% HER3MS17B05 17B057.55E−10  [4.5E−10, 8.1E−10]   87% HER3MS00052 18G11 35GS 17B05 9GSALB11  7.6E−10  [6.6E−10, 8.8E−10]   97% HER3MS34C07 34C07 8.15E−10[2.16E−10, 9.8E−10] 65.20% HER3MS18G11 18G11 no comp NA no comp

Example 15 Analysis of the HRG Induced Signaling Blocking Capacity ofthe Multivalent Parental Nanobodies with ALB11 HLE in Cellular Assays

To determine the potency of multivalent Nanobodies in inhibiting ligandinduced HER3 phosphorylation and EGFR/Her3 and Her2/Her3transphosphorylation, Nanobodies were tested in cell based assays asdescribed below. Inhibition of ligand induced pHER3 (phosphoHER3) inMCF-7 cells (cell based electrochemiluminescence assay (ECLA)): MCF-7cells (ATCC HTB 22) were serum starved and pre-incubated with Nanobodies(serial dilutions, starting concentration 666 nM or 167 nM) inserum-free media for 60 min at 37° C., 5% CO2. Cells were stimulatedwith 50 ng/ml HRG1-β1 EGF domain (R&D Systems, #396-HB) for 10 min,supernatants discarded and cells lysed in cold NP-40 lysis buffer (1%NP-40, 20 mM Tris, pH8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, proteaseinhibitor cocktail set III (Calbiochem), phosphatase inhibitor cocktailset II (Calbiochem)). MA6000 96 well plates (MSD, # L15XB) were blockedwith 3% block A (MSD) in PBS, pH7.4, 0.05% Tween20 and coated with HER3specific capture antibody (R&D Systems, # MAB3481). Cell lysates wereadded and incubated for 2 h at room temperature (RT). Biotinylatedanti-phospho Tyrosine antibody (R&D Systems, # BAM1676) and sulfo tagstreptavidin reagent (MSD, #R32AD) were used for detection (Table C-14).Inhibition of EGFR/HER3 and HER2/HER3 transphosphorylation: MDA MB468(ATCC HTB 132) and CHO HER2/HER3 cells were serum starved and treated asdescribed above (MDA MB468 stimulation with 50 ng/ml HRG 1-β1 EGFdomain; CHO HER2/HER3 cells stimulation with 100 ng/ml HRG1-β1 EGFdomain). Cell lysates were tested in pHER3 ECLA (Table C-14).

Multivalent parental Nanobodies with Alb11 potently inhibited HRG1-β1induced HER3 signaling in MCF-7 cells, as well as cells expressingHER2/HER3 (CHO HER2/HER3) and cells expressing EGFR/HER3 (MDA-MB468).

TABLE C-14 Inhibition of HRG induced HER3 phosphorylation by multivalentparental Nanobodies with Alb11 HLE MCF-7, CHO HER2/HER3, MDA468, HRG1-β1HRG1-β1 HRG1-β1 construct Nb1 linker Nb2 linker ALB11 IC50 (M) IC50 (M)IC50 (M) HER3MS00022 21F06 35GS 21F06 9GS ALB11  5.90E−11 1.84E−091.54E−10 HER3MS00023 21F06 35GS 34C07 9GS ALB11  1.82E−10 7.06E−101.51E−11 HER3MS00026 34C07 35GS 21F06 9GS ALB11 8.856E−10 2.163E−09 2.367E−10  HER3MS00028 34C07 35GS 4C07 9GS ALB11  2.24E−09 2.06E−095.39E−10 HER3MS00030 4C07 35GS 21F06 9GS ALB11  3.18E−10 4.50E−094.69E−10 HER3MS00032 4C07 35GS 4C07 9GS ALB11  4.89E−10 4.78E−101.04E−10 HER3MS00034 21F06 35GS 18G11 9GS ALB11  7.24E−10 1.29E−089.80E−10 HER3MS00035 4C07 35GS 18G11 9GS ALB11  4.16E−10 6.88E−108.61E−11 HER3MS00037 18G11 35GS 18G11 9GS ALB11  7.29E−09 5.95E−091.15E−09 HER3MS00038 18G11 35GS 21F06 9GS ALB11  2.62E−10 3.63E−094.65E−10 HER3MS00039 18G11 35GS 4C07 9GS ALB11  2.26E−09 2.40E−091.96E−10 PHER3MS00051 34C07 35GS 17B05 9GS ALB11 1.401E−08 n.d. n.d.PHER3MS00052 18G11 35GS 17B05 9GS ALB11 7.446E−09 n.d. n.d. PHER3MS0005417B05 35GS 34C07 9GS ALB11 2.673E−09 n.d. n.d. PHER3MS00055 4C07 35GS17B05 9GS ALB11  2.69E−09 n.d. n.d. PHER3MS00056 17B05 35GS 17B05 9GSALB11 2.579E−09 n.d. n.d. PHER3MS00057 17B05 35GS 4C07 9GS ALB11 2.01E−09 n.d. n.d. PHER3MS00058 17B05 35GS 18G11 9GS ALB11 4.434E−09n.d. n.d. PHER3MS00060 21F06 35GS 17B05 9GS ALB11 1.513E−09 n.d. n.d.PHER3MS00061 17B05 35GS 21F06 9GS ALB11 3.041E−09 n.d. n.d. PHER3MS0006218G11 35GS 3407 9GS ALB11 1.124E−08 n.d. n.d. PHER3MS00063 34C07 35GS18G11 9GS ALB11 1.225E−08 n.d. n.d. HER3MS00110 21F06 9GS ALB11 9GS17B05 1.569E−09 n.d. n.d. HER3MS00111 17B05 9GS ALB11 9GS 17B051.523E−09 n.d. n.d. HER3MS00112 21F06 9GS ALB11 9GS 18G11  3.24E−10 n.d.n.d. HER3MS00113 18G11 9GS ALB11 9GS 21F06 8.608E−10 n.d. n.d.HER3MS00114 17B05 9GS ALB11 9GS 4C07 2.994E−09 n.d. n.d. HER3MS001154C07 9GS ALB11 9GS 17B05 1.939E−09 n.d. n.d. HER3MS00116 21F06 35GSALB11 35GS 17B05 6.238E−10 n.d. n.d. HER3MS00117 21F06 35GS ALB11 35GS18G11 6.667E−10 n.d. n.d. n.d. = not determined

Example 16 Analysis of the EGFR-HER3 Heterodimerization BlockingCapacity of the Multivalent Parental Nanobodies with ALB11 HLE inCellular Assays

Multivalent parental Nanobodies containing 17B05 as one building block,Alb11, and a second HER3 Nanobody (04C07, 18G11, 21F06, 34C07, or 17B05)were tested for their ability to block EGFR ligand induced Her3phosphorylation. All 17B08 containing multivalent parental Nanobodiespotently blocked TGFα induced pHER3 in CHO EGFR/HER3 cells asdemonstrated for monovalent 17B05.

CHO EGFR/HER3 cells were serum starved and pre-incubated with Nanobodiesor EGFR antibody cetuximab (serial dilutions, starting concentration166.7 nM) in serum-free media for 60 min at 37° C., 5% CO₂. Cells werestimulated with 100 ng/ml rhTGFα (R&D Systems, #239-A) for 10 min,supernatants discarded and cells lysed in cold NP-40 lysis buffer (1%NP-40, 20 mM Tris, pH8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, proteaseinhibitor cocktail set III (Calbiochem), phosphatase inhibitor cocktailset II (Calbiochem)). MA6000 96 well plates (MSD, # L15XB) were blockedwith 3% block A (MSD) in PBS, pH7.4, 0.05% Tween20 and coated with HER3specific capture antibody (R&D Systems, # MAB3481). Cell lysates wereadded and incubated for 2 h at room temperature (RT). Biotinylatedanti-phospho Tyrosine antibody (R&D Systems, # BAM1676) and sulfo tagstreptavidin reagent (MSD, #R32AD) were used for detection (Table C-15).

TABLE C-15 Inhibition of TGFα induced HER3 phosphorylation bymultivalent parental Nanobodies with Alb11 HLE and 17B05 CHO EGFR/HER3,TGFα construct Nb1 linker Nb2 linker ALB11 IC50 (M) PHER3MS00051 34C0735GS 17B05 9GS ALB11 3.967^(E)−09 PHER3MS00052 18G11 35GS 17B05 9GSALB11 5.624^(E)−09 PHER3MS00054 17B05 35GS 34C07 9GS ALB11 2.316^(E)−09PHER3MS00055 4C07 35GS 17B05 9GS ALB11 7.899^(E)−09 PHER3MS00056 17B0535GS 17B05 9GS ALB11 1.459^(E)−09 PHER3MS00057 17B05 35GS 4C07 9GS ALB116.416^(E)−09 PHER3MS00058 17B05 35GS 18G11 9GS ALB11 5.782^(E)−09PHER3MS00060 21F06 35GS 17B05 9GS ALB11  1.08^(E)−09 PHER3MS00061 17B0535GS 21F06 9GS ALB11 2.586^(E)−09 HER3MS00110 21F06 9GS ALB11 9GS 17B05 1.41^(E)−09 HER3MS00111 17B05 9GS ALB11 9GS 21F06  1.61^(E)−09HER3MS00114 17B05 9GS ALB11 9GS 04C07 3.332^(E)−09 HER3MS00115 4C07 9GSALB11 9GS 17B05 8.373^(E)−09 HER3MS00116 21F06 35GS ALB11 35GS 17B051.772^(E)−09 control Mab-1 <50% <50% = less than 50% inhibition

Example 17 Induction of HER3 Internalization by the Multivalent ParentalNanobodies with ALB11 HLE

The effect of HER3-specific Nanobodies on HER3 internalization wasassessed using MCF-7 and MALME-3M cells. Cells were seeded in 6 wellplates at a concentration of 10⁵ cells/ml and incubated in a cellincubator at 37° C. for 2 days. Then, 100 nM Nanobody was added with orwithout 5 μM HSA (Sigma, A8763) and cells were 2 h incubated at 4° C. or37° C. Wells with control MAb1 and wells without any Nanobody or MAbwere taken along as positive and negative controls respectively. Afterwashing the cells, they were harvested and stained with anti-HER3 (R&Dsystems, #AF234) and donkey anti-goat-PE (Jackson ImmunoResearchLaboratories). The samples were measured using a FACS Array (BectonDickinson) and the percentage internalization was determined bycomparing the obtained signal of HER3 surface expression in 37° C.conditions with that in the negative control condition at 4° C. None ofthe monovalent Nanobodies was able to induce HER3 internalization,whereas bivalent 17B05 induced 17.8% internalization. Bivalent 18G11,34C07, 4C07 and 21F06 induced 60-70% internalization, which is slightlyhigher than the positive control MAb1 (57% internalization). Inductionof HER3 internalization by biparatopic constructs was dependent on theirrespective compositions. No internalization was observed when 21F06 waspresent in the construct. In constructs without 21F06, internalization(40-70%) was only observed when 17B05 or 04C07 was located at theN-terminal position. Identical results were obtained in both cell lines.

Example 18 Sequence Optimization

Nanobodies 4C07, 17B05, 21F06, 18G11 and 34C07 were taken further forsequence optimization. This is a process in which parental Nanobodysequences are mutated to yield Nanobody sequences that are moreidentical to human VH3-JH germline consensus sequences. Specific aminoacids, with the exception of the so-called hallmark residues, in the FRsthat differ between the Nanobody and the human VH3-JH germline consensusare altered to the human counterpart in such a way that the proteinstructure, activity and stability are kept intact. The parental Nanobodyamino acid sequence is also aligned to the llama IGHV germline aminoacid sequence of the Nanobody (identified as the top hit from a BlastPanalysis of the Nanobody against the llama IGHV germlines), and incertain cases mutations towards the llama germline are introduced toincrease the stability of the Nanobody, which is defined asllamanisation. In addition potential sites for Post TranslationalModifications (PTMs as deamidation, isomerisation, methionine oxidation)are changed to guarantee chemical stability. The analysis occurred intwo rounds, in a first round single mutations and combined mutationsupon the basic variants were evaluated. Based upon these results,acceptable substitutions are combined in second round variants which arefurther characterized.

Six amino acid residues in 04C07 can be substituted for humanizationpurposes. In the sequence optimization process, four 04C07 versions (abasic version and 3 additional variants) were constructed. The basicvariant (HER3MS0042) contains 4 substitutions: A14P, A74S, K83R andQ108L. In addition to these changes, the A46E and L93A substitutionswere introduced and investigated in additional variants. The constructswere expressed in E. coli and purified by IMAC and desalting.

The purified molecules were evaluated for their HRG blocking capacityusing alphascreen. Also thermal stability of the variants was tested ina thermal shift assay using the Lightcycler (Roche). In this assay theNanobody variants are incubated at different pH's in the presence ofsypro orange and a temperature gradient is applied. When the Nanobodiesstart denaturating, sypro orange binds and the measured fluorescenceincreases suddenly. A melting temperature can be determined for acertain pH. Results are summarized in C-16.

TABLE C-16 results of the first round sequence optimization variants of04C07 IC50 HRG Tm at competition pH 7 ID Mutation(s) alphascreen (M) (°C.) HER3MS04C07 parental 2.7E−10-6.9E−10 58.55 HER3MS0042 A14P, A74S,K83R,  3.1E−10 60.3 (basic) Q108L HER3MS0043 basic + A46E 1.02E−9 66.5HER3MS0044 basic + L93A 1.00E−9 62 HER3MS0045 basic + A46E + L93A4.91E−9 66.9

The basic variant had a similar potency in the HRG blocking alphascreenas the parental 04C07 Nanobody, meaning that the mutations on positions14, 74, 84 and 108 were accepted. A 3- to 4-fold lower potency wasobserved in variants HER3MS0043 (A46E) and HER3MS0044 (L93A), whichincreases to a 18-fold potency drop if they were combined. All variantshad a positive influence on stability as higher Tm values were obtainedthan for the parental 04C07 Nanobody. Analysis of post translationalmodification sites indicated deamidation at position N101 (22-29%deamidation after 4 weeks storage at 40° C. and 9-14% deamidation after3 days storage at pH9 and 25° C.). A library of N101X was made,transfected in E. coli and Nanobody containing periplasmic lysates weretested for their HRG blocking capacity in a competition FMAT as well asfor their off rate on recombinant HER3. All tested variants gave 99-100%blocking and a 2-3 fold higher off rate compared to the parental 04C07Nanobody. The variants N101G and N101S were selected for furthercharacterization. Pyroglutamate formation was 6.6% after 4 weeks storageat 40° C., which was acceptable and did not require an amino acidsubstitution at position 1. Four variants were made in the second roundsequence optimization. They were combinations of the basic variant withsubstitutions A46E, N101G or N101S (Table C-17). Introduction of thesubstitution A46E resulted in a significant potency drop in the HRGcompetition alphascreen and pHER3 blocking assay in MCF7 cells but onthe other hand increased stability as a Tm increase of 4.5° C. wasobserved. These results were in agreement with first round sequenceoptimization results. Mutation of N101 to G or S gave a 5- to 6-foldpotency drop and a decrease in Tm of 1.5-2° C. Variants HER3MS00129 andHER3MS00130 were selected for further characterization as formattingcould mask their lower potency in a monovalent format. The percentageframework identity in the framework region for HER3MS00129 is 95.5% andfor HER3MS00130 94.4%, based on the AbM definition.

TABLE C-17 results of the second round sequence optimization variants of04C07 IC50 HRG IC50 pHER3 competition HRG1-β1 TM alphascreen stimulatedpH 7 ID Mutations (M) MCF7 (M) (° C.) HER3MS04C07 parental 1.8E−106.25E−09 58.55 HER3MS0042 A14P, A74S, 3.1E−10 6.805E−9  60.3 (basic)K83R, Q108L HER3MS00129 basic + A46E + 5.1E−9  1.21E−07 63.1 N101GHER3MS00130 basic + N101G 8.8E−10 9.38E−09 56.5 HER3MS00131 basic +A46E + 5.8E−9  2.26E−07 63.5 N101S HER3MS00132 basic + N101S 1.0E−9 1.31E−08 56.9

Five amino acid residues in 21F06 can be substituted forhumanization/llamanization purposes and five amino acid substitutionscan be analysed for chemical stability purposes. In the sequenceoptimization process, four 21F06 versions (a basic version and 3additional variants) were constructed. The basic variant (HER3MS0046)contains 4 substitutions: A14P, A74S, K83R and Q108L. In addition tothese changes, the G40A and D44E substitutions were introduced andinvestigated in additional variants. The constructs were expressed in E.coli and purified by IMAC and desalting. The purified molecules wereevaluated for their HRG blocking capacity using alphascreen. Alsothermal stability of the variants was tested in a thermal shift assayusing sypro orange and applying a temperature gradient. When theNanobodies start denaturating, sypro orange binds and the measuredfluorescence increases suddenly. A melting temperature can be determinedfor a certain pH. Results are summarized in Table C-18.

TABLE C-18 Results of 21F06 humanization variants in the first roundsequence optimization IC50 HRG IC50 pHER3 competition HRG1-β1 Tm atalphascreen stimulated pH 7 ID Mutations (M) MCF7 (M) (° C.) HER3MS21F06parental 2.5E−10 8.74E−9 67.3 HER3MS0046 A14P, A74S, 2.8E−10 NA 67.4basic K83R, Q108L HER3MS0047 basic + G40A 2.1E−10 NA 71.1 HER3MS0048basic + D44E 2.7E−10 NA 67.8 HER3MS0049 basic + G40A + 2.5E−10 2.79E−970.6 D44E

All tested variants had a similar potency in the HRG blockingalphascreen as the parental 21F06 Nanobody, meaning that the mutationson positions 14, 40, 44, 74, 84 and 108 were accepted. The G40Asubstitution resulted in an increased Tm value, suggesting that it had apositive effect on the stability of the Nanbody. To deal with 2potential isomerisation sites and one Met oxidation site, libraries weremade where the amino acids D54, G55, M100b, D101 and S102 wererandomized. The libraries were transformed in TG1 and individualcolonies were picked and grown in 96-well plates. Periplasmic extractswere prepared, sequenced and screened. All tested variants as well as21F06 parental gave complete block in a HRG competition FMAT experimentand off rate analysis also could not discriminate between these variantsand 21F06 parental (6.2E-4-8.3E-4). Some variants were selected (basedon similar characteristics of original and substituted amino acid) topurify and test for their thermal stability. Results are shown in C-19.

TABLE C-19 Tm results of 21F06 chemical stability variants in the firstround sequence optimization ID Mutations Tm at pH 7 (° C.) HER3MS21F06Parental (P) 64   HER3MS00088 P + D54Y − Q108L  60.7/61.06 HER3MS00089P + D54E − Q108L 61.1/61.9 HER3MS00090 P + G55A − Q108L 54.8 HER3MS00091P + M100bF − Q108L 56.1/56.8 HER3MS00092 P + M100bY − Q108L 56.9/57.3HER3MS00093 P + M100bL − Q108L 60.7 HER3MS00094 P + D101Q − Q108L 62.3HER3MS00095 P + D101E − Q108L 60.3/60.7 HER3MS00096 P + S102D − Q108L50.3 HER3MS00097 P + S102E − Q108L 55.7 HER3MS00098 P + S102T − Q108L65.2

Analysis of post translational modification sites indicated 14%pyroglutamate after 4 weeks storage at 40° C. and no substitution wasintroduced at position 1. Thirteen percent of the mono-oxidized materialwas observed after 3 h treatment with 10 mM H2O₂ at room temperature.Substitution of M100b was required and mutation M100bL was selectedbased on screening data and physical similarity between M and L. Storageof parental 21F06 under forced deamidation conditions (4 weeks at 40° C.or 3 days at pH9 and 25° C.) induced 20% deamidation at position N73.However, the percentage deamidation was only 3.2 after 3 weeks storageat 25° C. Analysis of a 21F06 variant containing the basic mutation A74Sshowed a slightly higher percentage of deamidation: 37.1% after storagein forced deamidation conditions (3 days at pH9 and 25° C.) and 4.9%after 3 weeks storage at 25° C. It was concluded to keep A74S forfurther characterization in a second round of sequence optimization asthis increased the percentage of framework identity compared to humanantibodies and deamidation could be kept under control during DSP/USPsince only a deamidated fraction of <5% was observed in the 25° C.conditions. Finally, 12.9% deamidation was observed of D54 and mutationD54E was selected based on screening data and physical similaritybetween D and E.

The second round 21F06 sequence optimization variant HER3MS00122 wasmade which combined the substitutions A14P, G40A, D44E, A74S, K83R andQ108L. This variant and parental 21F06 gave a similar IC50 in a HRGalphascreen competition assay as well as in the pHER3 blocking assayusing MCF7 cells and showed an identical Tm value. 21F06 variantHER3MS00122 has 91% framework identity in the framework region accordingto the AbM definition.

Based on these results, HER3MS00122 was selected as the final sequenceoptimized 21F06 Nanobody.

Five amino acid residues in 18G11 can be substituted for humanizationpurposes and four amino acid substitutions can be analysed for chemicalstability purposes. In this case the basic variant was made, containingmutations K₈₃R and Q108L. Then, a mini-library of the basic variant with2⁶=64 permutations at positions 44, 61, 64, 74, 75 and 77 wasconstructed. Mutation H93A was not investigated since this mutation isclose to CDR3 which increased the chance that humanization would affectthe potency of the Nanobody.

The libraries were transformed in TG1 and individual colonies werepicked and grown in 96 well plates. Periplasmic extracts were prepared,sequenced and screened for competition with biotinylated parental 18G11to bind recombinant HER3-Fc. The parental 18G11 Nanobody and its basicvariant HER3MS0050 showed identical potency in alphascreen (IC50 3.2E-9M) and an identical Tm of 72° C. The substitution W75K was not toleratedas a total loss in HER3 binding was observed. Thirty three uniquecompetitors were tested for off rate on recombinant HER3-ECD. Mostclones were within a 2-fold higher k_(d) range compared to parental18G11 (k_(d) 6.2-7.7E-4 s⁻¹), only three variants showed a 2-3 foldhigher off rate. The results suggested that substitutions E64K, T74S,A77T, K83R, Q108L had no effect on the potency or stability of 18G11,whereas a 28% reduction in potency was observed in presence ofsubstitution R44Q. However, the R44Q substitution increased the Tmvalues with around 4° C. suggesting that the stability of the moleculesimproved.

Analysis of post translational modification sites revealed 12.3%pyroglutamate formation at position 1 after 4 weeks storage at 40° C.but this level did not require substitution of E1. No isomerizationvariants at position 95 could be detected after 4 weeks storage at 40°C. and significant oxidation was only observed at amino acids M99 andM102 (30-40% mono-oxidized variant after forced oxidation using 10 mMH₂O₂ during 3 h at room temperature). Two substitution libraries weremade to deal with these Met oxidation sites (M99X and M102X). Thelibraries were transformed in TG1 and individual colonies were pickedand grown in 96 well plates. Periplasmic extracts were prepared,sequenced and screened for competition with biotinylated parental 18G11to bind recombinant HER3-Fc. Subsequently, 18 unique competitors weretested in off rate screening on recombinant HER3-ECD. Sixteen clonesshowed identical k_(d) values as parental 18G11 (6.10E-4 s−1). Sevenvariants were selected for purification and further characterization fortheir capacity to block pHER3 signaling in MCF7 cells and theirstability using the thermal shift assay. The results are shown in TableC-20. The substitutions M99L and M102L were selected to test in a secondround sequence optimization based on potency, stability and similarphysical properties of leucine and methionine.

TABLE C-20 Results of 18G11 variants in the first round sequenceoptimization Nanobody tested as peri purified Nanobody tested 18G11 IC50pHER3 competition blocking HRG Tm at alphascreen Off rate stimulated pH7 ID mutation (% inhibition) (s⁻¹) MCF7 (M) (° C.) 18G11 parental91-93.6%    6.2-7.7E−4     2.912E−8 69.9 HER3MS00069 M99L  83% 8.1E−4 5.27E−8 71.88 HER3MS00070 M99I 82.2% 6.4E−4 3.778E−8 71.06 HER3MS00071M99V 80.9% 8.4E−4 7.346E−8 71.06 HER3MS00068 M99G 84.4% 1.0E−3 8.881E−870.64 HER3MS00072 M102L 88.0% 8.2E−4 5.764E−8 70.63 HER3MS00073 M102D84.4% 8.3E−4 8.747E−8 62.73 HER3MS00074 M102E 82.7% 4.9E−4 6.304E−8 66.1In the second round 18G11 sequence optimization, two variants were madewhich combined the selected substitutions for humanization and chemicalstability purposes. The only difference between both variants was thepresence/absence of R44Q. Both variants showed an increase of around 6°C. in Tm value compared to the parental Nanobody (Table C-21). Finally,the 18G11 variant HER3MS00211 was selected as absence of the R44Qmutation resulted in a slightly better potency. The obtained percentageframework identity in the framework region was 87.6 according to the AbMdefinition (see Antibody Engineering, Vol 2 by Kontermann & Diibel(Eds), Springer Verlag Heidelberg Berlin, 2010).

TABLE C-21 Results of 18G11 variants in the second round sequenceoptimization IC50 pHER3 IC50 18G11 blocking HRG Tm at competitionstimulated pH ID Mutations alphascreen (M) Off-rate (s⁻¹) MCF7 (M) (°C.) HER3MS18G11 — 4.4E−09 4.3E−4-4.7E−4 1.09E−8-4.61E−8 69.9 parentalHER3MS00125 R44Q, S62D, E64K, T74S, 1.4E−08 1.2E−03 76.0 A77T, K83R,M99L, M102L and Q108L HER3MS00211 E64K, T74S, A77T, M99L, 5.4E−097.2E−04 1.79E−8-2.0E−8  75.6 K83R, M102L and Q108L

Seven amino acid residues in 34C07 can be substituted for humanizationpurposes. In this case the basic variant was made, containing mutationsK83R and Q108L. Then, a mini-library of the basic variant with 7additional unique mutations spread over 6 variable positions (2⁵×3=96permutations) was constructed. Mutation H93A was not investigated sincethis mutation is close to CDR3 which increased the chance thathumanization would affect the potency of the Nanobody. The librarieswere transformed in TG1 and individual colonies were picked and grown in96 well plates. Periplasmic extracts were prepared, sequenced andscreened for competition with biotinylated parental 34C07 to bindrecombinant HER3-Fc. Twelve unique competitors were tested for off rateon recombinant HER3-ECD. Most clones were within a 2-fold higher k_(d)range compared to parental 34C07 (k_(d) 3.7E-4-3.9E-4 s⁻¹). The mutationW75K showed less than 40% inhibition in the 34C07 competitionalphascreen and a 95-fold increase in off rate compared to parental34C07. The substitutions G19R, V23A, V71R, A74S, V84P and V84A did notaffect binding to HER3.

Analysis of post translational modification sites revealed 42%pyroglutamate formation after 4 weeks storage at 40° C. Therefore, theeffect of the substitution E1D on the Nanobody potency was analysed inthe second round sequence optimization. No oxidation (10 mM H₂O₂ during3 h at room temperature), deamidation (4 weeks at 40° C.) or Aspisomerization (4 weeks at 40° C.) were identified so there was no needto substitute additional amino acids for chemical stability purposes.

Four constructs were made in the second round sequence optimization.They all contained the substitutions G19R, V23A, V71R, A74S, K83R andQ108L but were different for E1D, V84A and V84P (Table C-22). Theconstructs were expressed in E. coli and purified by IMAC and desalting.The purified molecules were characterized using the 34C07 competitionalphascreen, off rate analysis on HER3-ECD and pHER3 blocking assay inHRG stimulated MCF7 cells. Results are summarized in Table C-22.

TABLE C-22 Results of 34C07 variants in the second round sequenceoptimization IC50 34C07 IC50 pHER3 competition blocking ID Mutationsalphascreen (M) Off-rate (s⁻¹) MCF7 (M) Tm (° C.) HER3MS34C07 — 5.5E−93.0E−4-3.1E−4 2.49E−8 73.9 parental HER3MS00123 K83R + Q108L + G19R +7.6E−9 4.6E−4 3.09E−8 76.4 V23A + V71R + A74S + V84P HER3MS00124 K83R +Q108L + G19R + 7.1E−9 4.5E−4 3.05E−8 76.2 V23A + V71R + A74S + V84AHER3MS00127 K83R + Q108L + E1D + 8.5E−9 4.6E−4 3.88E−8 75.6 G19R +V23A + V71R + A74S + V84P HER3MS00128 K83R + Q108L + E1D + 6.7E−9 4.6E−44.05E−8 75.2 G19R + V23A + V71R + A74S + V84A

It was confirmed that the investigated substitutions did not influencethe binding significantly. In addition, pHER3 blocking, off rate and Tmwere similar for all tested variants and the parental 34C07 Nanobody.The final variant became HER3MS00123 and HER3MS00127 (containing the E1Dmutation in case the Nanobody is N-terminally located in the multivalentconstruct). The percentage framework identity in the framework regionsfor HER3MS00123 is 89.9% and for HER3MS00127 is 88.8% based on the AbMdefinition.

Six amino acid residues in 17B05 can be substituted for humanizationpurposes and two amino acid substitutions can be analysed for chemicalstability purposes. In this case the basic variant was made, containingmutations A74S, K83R and Q108L. Then, nine constructs were madecontaining the basic mutations and combinations of the three additionalmutations which were investigated (173N, F79Y and R93A).

The constructs were expressed in E. coli and purified by IMAC anddesalting. The purified molecules were tested for their capacity toblock HRG binding to HER3-Fc using alphascreen, off rate on HER3-ECD andstability using the thermal shift assay. Results are summarized in C-23.

TABLE C-23 Results of 17B05 variants in the first round sequenceoptimization IC50 HRG competition Tm at pH 7 ID Mutations alphascreen(M) Off-rate (s⁻¹) (° C.) HER3MS17B05 — 3.4E−10-9.3E−10 1.8E−4-2.0E−469.8 parental HER3MS0076 A74S + N83R + 7.88E−10 1.7E−4 73.6 basicvariant Q108L HER3MS0077 A74S + N83R + 7.53E−10 NA 72.3 Q108L + I73NHER3MS0078 A74S + N83R + 8.11E−10 NA 74.4 Q108L + F79Y HER3MS0079 A74S +N83R + 3.08E−09 NA 71.5 Q108L + R93A HER3MS0080 A74S + N83R + 7.31E−101.8E−4 73.6 Q108L + I73N + F79Y HER3MS0081 A74S + N83R + 5.89E−09 NA69.4 Q108L + I73N + R93A HER3MS0082 A74S + N83R + 4.05E−09 NA 73.2Q108L + F79Y + R93A HER3MS0083 A74S + N83R + 4.95E−09 8.4E−4 71.1Q108L + I73N + F79Y + R93A HER3MS0084 A74S + N83R + 9.4E−10 1.8E−4 NAQ108L +S84P HER3MS0085 A74S + N83R + 7.6E−10-9.4E−10 1.9E−4 74.8 Q108L +S84A

At least a 9-fold reduction in potency was observed in the HRG blockingalphascreen when substitution R93A was introduced in the construct,whereas other substitutions resulted in a similar IC50 compared toparental 17B05. This observation was confirmed by the off rate data.Most mutations also had a small positive effect in Tm value.

Analysis of post translational modification sites revealed 25%pyroglutamate formation after 4 weeks storage at 40° C. Therefore, theeffect of the substitution E1D on the Nanobody potency was analysed inthe second round sequence optimization. No oxidation (10 mM H₂O₂ during3 h at room temperature) or Asn deamidation (4 weeks at 40° C.) wereidentified so there was no need to substitute additional amino acids forchemical stability purposes.

Four constructs were made in the second round sequence optimization.They all contained the substitutions 173N, F79Y, A74S, K83R and Q108Lbut were different for E1D, S84A and S84P (Table C-24). The constructswere expressed in E. coli and purified by IMAC and desalting. Thepurified molecules were characterized using the HRG competitionalphascreen on HER3-Fc, off rate analysis on HER3-ECD and pHER3 blockingassay in MCF7 cells. Results are summarized in Table C-24.

TABLE C-24 Results 17B05 variants in the second round sequenceoptimization IC50 pHER3 blocking HRG HRG competition Off-rate stimulatedTm at pH 7 ID Mutations alphascreen (M) (s⁻¹) MCF7 (M) (° C.)HER3MS17B05 parental 7.8E−10 1.8E−4-2.0E−4 4.52E−9 69.8 HER3MS0118A74S + N83R + Q108L + 6.0E−10 1.8E−4 2.69E−9 74.4 I73N + F79Y + S84A +R93A HER3MS0119 A74S + N83R + Q108L + 7.9E−10 1.8E−4 1.70E−9 76.9 I73N +F79Y + S84P + R93A HER3MS0120 A74S + N83R + Q108L + 7.5E−10 1.9E−44.28E−9 73.9 E1D + I73N + F79Y + S84A + R93A HER3MS0121 A74S + N83R +Q108L + 5.5E−10 1.8E−4 3.53E−9 76.4 E1D + I73N + F79Y + S84P + R93A

It was confirmed that the investigated substitutions did not influencethe binding and HRG competition significantly. In addition, IC50s forpHER3 blocking were similar for all tested variants and the parental17B05 Nanobody. Introduction of S84P had a positive effect on Tm. Thefinal variant became HER3MS00119 and HER3MS00121 (containing the E1Dmutation in case the Nanobody is N-terminally located in the multivalentconstruct). The percentage framework identity in the framework regionsfor HER3MS00119 is 89.9% and for HER3MS00121 is 88.8% based on the AbMdefinition.

Example 19 Generation and Expression Level Analysis of the MultivalentSequence Optimized HER3-Specific Nanobodies with HLE

Combinations were made of sequence optimized Nanobodies blocking HRGbinding to HER3 (21F06, 04C07 and 17B05), a Nanobody blocking HER3heterodimerization (17B05) and a Nanobody binding to domain II of HER3(18G11). The ALB half life extension was located in the middle(Nb1-ALB-Nb2) or at the C-terminal position (Nb1-Nb2-ALB). The differentbuilding blocks were connected with two 9GS linkers or two 35GS linkers.The constructs were produced in Pichia pastoris as tagless proteins andpurified via MEP hypercell or Protein A affinity chromatography,followed by desalting. A copy number screen was performed and expressionyields were predicted for the clones with the highest copy number basedon small scale cultures (Table C-25). Little impact of position ofNanobody building blocks and linker length on predicted yields wasobserved.

TABLE C-25 Results of expression level analysis of purified multivalentsequence optimized anti-HER3 Nanobodies with ALB11 HLE Predicted yieldin Nb2 or Nb2 or fermentor ID Nb1 linker HLE linker HLE (g/l)HER3MS00135 17B05 SO 9GS ALB11 9GS 21F06 SO 1.5 HER3MS00136 17B05 SO35GS ALB11 35GS 21F06 SO >1 HER3MS00137 17B05 SO 9GS 21F06 SO 9GS ALB111.5 HER3MS00138 17B05 SO 35GS 21F06 SO 35GS ALB11 0.5-1 HER3MS0013917B05 SO 9GS ALB11 9GS 34C07 SO 1.5 HER3MS00140 17B05 SO 35GS ALB11 35GS34C07 SO >1 HER3MS00141 17B05 SO 9GS 34C07 SO 9GS ALB11 1.5 HER3MS0014217B05 SO 35GS 34C07 SO 35GS ALB11 >1 HER3MS00143 17B05 SO 9GS ALB11 9GS18G11 SO 1.5 HER3MS00144 17B05 SO 35GS ALB11 35GS 18G11 SO 0.5-1HER3MS00145 17B05 SO 9GS 18G11 SO 9GS ALB11 1.5 HER3MS00146 17B05 SO35GS 18G11 SO 35GS ALB11 0.5-1 HER3MS00209 17B05 SO 9GS ALB11 NA NA 1.5HER3MS00212 17B05 SO 9GS ALB11 9GS 18G11 SO 1 HER3MS00159 18G11 SO 35GSALB11 35GS 04C07 SO 1.5 HER3MS00160 18G11 SO 35GS ALB11 35GS 04C07 SO0.5 HER3MS00161 18G11 SO 35GS 04C07 SO 35GS ALB11 >1 HER3MS00162 18G11SO 35GS 04C07 SO 35GS ALB11 >1 HER3MS00199 18G11 SO 9GS ALB11 9GS 21F06SO 0.5-1 HER3MS00200 18G11 SO 9GS 21F06 SO 9GS ALB11 0.5-1 HER3MS0020718G11 SO 9GS ALB11 9GS 17B05 SO 0.5-1 HER3MS00215 18G11 SO 9GS ALB11 9GS17B05 SO 0.5-1 HER3MS00201 21F06 SO 9GS ALB11 9GS 18G11 SO 0.5-1HER3MS00202 21F06 SO 9GS 18G11 SO 9GS ALB11 0.5-1 HER3MS00210 21F06 SO9GS ALB11 9GS 18G11 SO 0.5 HER3MS00214 21F06 SO 9GS ALB11 9GS 18G11 SO0.5 HER3MS00152 34C07 SO 35GS ALB11 35GS 04C07 SO 0.5-1 HER3MS0015334C07 SO 35GS 04C07 SO 35GS ALB11 0.5-1 HER3MS00154 34C07 SO 35GS 04C07SO 35GS ALB11 0.5-1 HER3MS00147 04C07 SO 35GS ALB11 35GS 34C07 SO 1.5HER3MS00149 04C07 SO 35GS 34C07 SO 35GS ALB11 >1 HER3MS00155 04C07 SO35GS ALB11 35GS 18G11 SO 1.5 HER3MS00157 04C07 SO 35GS 18G11 SO 35GSALB11 >1 HER3MS00208 04C07 SO 9GS ALB11 9GS 18G11 SO 0.5 HER3MS0021304C07 SO 9GS ALB11 9GS 18G11 SO 0.5-1 HER3MS00148 04C07 SO 35GS ALB1135GS 34C07 SO 0.5-1 HER3MS00150 04C07 SO 35GS 34C07 SO 35GS ALB11 0.5-1HER3MS00151 04C07 SO 35GS ALB11 35GS 34C07 SO 0.5-1 HER3MS00156 04C07 SO35GS ALB11 35GS 18G11 SO 0.5-1 HER3MS00158 04C07 SO 35GS 18G11 SO 35GSALB11 0.5-1

Example 20 Induction of HER3 Internalization by the Multivalent SequenceOptimized Nanobodies with ALB11 HLE

The effect of HER3-specific multivalent sequence optimized Nanobodieswith HLE on HER3 internalization was assessed using MCF-7 and MALME-3Mcells as described in the previous examples. No internalization wasobserved when 21F06 was present in a multivalent construct. Inconstructs without 21F06, internalization (36-71%) was only observedwhen 17B05 or 04C07 was present in the N-terminal position. The results(Table C-26) were identical in both cell lines. The constructHER3MS00209 consists of sequence optimized 17B05 linked to ALB11 and didnot show internalization. These results were similar to those obtainedwith the parental multivalent constructs and indicated that the selectedmultivalent panel contained molecules with different mode of actions.Two control antibodies were also tested in the internalization assay andthey induced both internalization of HER3.

TABLE C-26 Percentage HER3 internalization induced by multivalentsequence optimized Nanobodies or control MAbs on MCF7 and MALME-3Mcells. % HER3 % HER3 Nb2 or internalization MCF7 internalization ID Nb1linker HLE linker Nb2 or HLE cells MALME-3M cells HER3MS00135 17B05 SO9GS ALB11 9GS 21F06 SO 0 0 HER3MS00137 17B05 SO 9GS 21F06 SO 9GS ALB11 5NA HER3MS00143 17B05 SO 9GS ALB11 9GS 18G11 SO 62-67 NA HER3MS0020718G11 SO 9GS ALB11 9GS 17B05 SO 4 9 HER3MS00208 4C07 SO 9GS ALB11 9GS18G11 SO 36.0 40.2 HER3MS00209 17B05 SO 9GS ALB11 NA NA 0-7 0-4HER3MS00210 21F06 SO 9GS ALB11 9GS 18G11 SO 0 0 HER3MS00212 17B05 SO 9GSALB11 9GS 18G11 SO 65-71 64-70 HER3MS00213 4C07 SO 9GS ALB11 9GS 18G11SO 48-53 53 HER3MS00214 21F06 SO 9GS ALB11 9GS 18G11 SO 0 0 HER3MS0021518G11 SO 9GS ALB11 9GS 17B05 SO 0-1 0-6 Control MAb1 NA NA NA NA NA51-60 53-69 Control MAb2 NA NA NA NA NA 49-61 50-63

Example 21 Inhibition of the HRG Induced pHER3 and Downstream Signalingby Multivalent Sequence Optimized Nanobodies with ALB11 HLE in CellularAssays

To determine the potency of multivalent sequence optimized Nanobodies ininhibiting ligand induced HER3 phosphorylation and EGFR/HER3 andHER2/HER3 transphosphorylation, Nanobodies were tested in cell basedassays as described below.

Inhibition of ligand induced pHER3 (phosphoHER3) in MCF-7 cells (cellbased electrochemiluminescence assay (ECLA)): MCF-7 cells (ATCC HTB 22)were serum starved and pre-incubated with Nanobodies (serial dilutions,starting concentration 666 nM or 167 nM) in serum-free media for 60 minat 37° C., 5% CO₂. Cells were stimulated with 50 ng/ml HRG1-β1 EGFdomain (R&D Systems, #396-HB) for 10 min, supernatants discarded andcells lysed in cold NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH8.0, 137mM NaCl, 10% glycerol, 2 mM EDTA, protease inhibitor cocktail set III(Calbiochem), phosphatase inhibitor cocktail set II (Calbiochem)).MA6000 96 well plates (MSD, # L15XB) were blocked with 3% block A (MSD)in PBS, pH7.4, 0.05% Tween20 and coated with HER3 specific captureantibody (R&D Systems, # MAB3481). Cell lysates were added and incubatedfor 2 h at room temperature (RT). Biotinylated anti-phospho Tyrosineantibody (R&D Systems, # BAM1676) and sulfo tag streptavidin reagent(MSD, #R32AD) were used for detection (Table C-27).

21.1 Inhibition of EGFR/HER3 and HER2/HER3 Transphosphorylation:

MDA MB468 (ATCC HTB 132) and CHO HER2/HER3 cells were serum starved andtreated as described above (MDA MB468 stimulation with 50 ng/ml HRG1-β1EGF domain; CHO HER2/HER3 cells stimulation with 100 ng/ml HRG1-β1 EGFdomain). Cell lysates were tested in pHER3ECLA (Table C-27).

Multivalent sequence optimized Nanobodies with Alb11 potently inhibitedHRG induced HER3 signaling in MCF-7 cells, as well as cells expressingHER2/HER3 (CHO HER2/HER3) or cells expressing EGFR/HER3 (MDA-MB468). Inaddition downstream signaling was potently inhibited. Alb11 did notabolish potency in the formats tested.

21.2 Inhibition of Downstream Signaling (pAkt/pERK1/2):

Inhibition of downstream signaling (pAkt/pERK1/2): Inhibition of ligandinduced pAKT and pERK (phosphoAkt, phosphor ERK) in MCF-7 cells (cellbased electrochemi-luminescence assay (ECLA)): MCF-7 cells (ATCC HTB 22)were serum starved and pre-incubated with Nanobodies (serial dilutions,starting concentration 666 nM or 167 nM) in serum-free media for 60 minat 37° C., 5% CO2. Cells were stimulated with 50 ng/ml HRG1-β1 EGFdomain (R&D Systems, #396-HB) for 10 min, supernatants discarded andcells lysed in cold NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH8.0, 137mM NaCl, 10% glycerol, 2 mM EDTA, protease inhibitor cocktail set III(Calbiochem), phosphatase inhibitor cocktail set II (Calbiochem)). Celllysates were tested in Phospho-Akt (Ser473) Whole Cell Lysate Kit (MSD,# K151CAD) and Phospho-ERK1/2 Whole Cell Lysate Kit (MSD, # K111DWD)according to manufacturer's instructions (Table C-28).

Formatted sequence optimized Nanobodies were able to potently blockHRG-induced downstream signalling of HER3.

TABLE C-27 Inhibition of HRG induced pHER3 MCF-7, HRG CHO HER2/HER3,MDA-MB 468, construct IC50 (M) HRG IC50 (M) HRG IC50 (M) HER3MS001351.21E−10 (n = 4) 1.42E−09 (n = 3) 1.54E−10 (n = 3) HER3MS00137 8.80E−104.71E−09 1.12E−10 HER3MS00139 3.49E−09 6.12E−09 HER3MS00141 4.35E−094.58E−09 HER3MS00147 5.98E−09 9.12E−09 HER3MS00149 3.54E−09 3.58E−09HER3MS00150 2.32E−09 5.56E−09 5.38E−10 HER3MS00209 1.17E−09 (n = 3)4.07E−09 (n = 3) 1.02E−09 (n = 3) HER3MS00212 5.33E−10 (n = 4) 4.76E−09(n = 3) 8.00E−10 (n = 3) HER3MS00213 5.21E−10 (n = 3) 4.22E−09 (n = 3)5.99E−10 (n = 3) HER3MS00214 1.56E−10 (n = 3) 1.45E−09 (n = 3) 9.84E−11(n = 3) HER3MS00215 1.59E−09 (n = 4) 5.43E−09 (n = 3) 1.60E−09 (n = 3)

TABLE C-28 Inhibition of HRG induced pAKt and pERK pAKT inhibition pERKinhibition Nanobody (IC50 M) (IC50 M) HER3MS00135 1.494E−10 2.206E−10HER3MS00209  8.55E−10 nd HER3MS00212  5.56E−10 4.544E−10 HER3MS002135.381E−10 nd HER3MS00214 1.147E−10 nd HER3MS00215 1.294E−09 3.811E−09

Example 22 Analysis of the HER1-HER3 Heterodimerization BlockingCapacity of the Multivalent Sequence Optimized Nanobodies with ALB11 HLEin Cellular Assays

To determine the potency of multivalent sequence optimized Nanobodies ininhibiting EGFR ligand induced HER3 phosphorylation Nanobodies weretested in cell based assays as described below.

Inhibition of EGFR ligand induced pHER3 (phosphoHER3) in CHO EGFR/HER3cells (cell based electrochemiluminescence assay (ECLA)): CHO EGFR/HER3were serum starved and pre-incubated with Nanobodies (serial dilutions,starting concentration 666 nM or 167 nM) in serum-free media for 60 minat 37° C., 5% CO₂. Cells were stimulated with 100 ng/ml rhTGFα (R&DSystems, #239-A), for 10 min, supernatants discarded and cells lysed incold NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH8.0, 137 mM NaCl, 10%glycerol, 2 mM EDTA, protease inhibitor cocktail set III (Calbiochem),phosphatase inhibitor cocktail set II (Calbiochem)). MA6000 96 wellplates (MSD, # L15XB) were blocked with 3% block A (MSD) in PBS, pH7.4,0.05% Tween20 and coated with HER3 specific capture antibody (R&DSystems, # MAB3481). Cell lysates were added and incubated for 2 h atroom temperature (RT). Biotinylated anti-phospho Tyrosine antibody (R&DSystems, # BAM1676) and sulfo tag streptavidin reagent (MSD, #R32AD)were used for detection (Table C-29). Multivalent sequence optimizedNanobodies containing 17B05 as building block potently inhibited EGFRligand induced HER3 phosphorylation. Partial inhibition was observedwith the multivalent Nanobody 214 (HER3MS00214), which does not include17B05 as a building block.

TABLE C-29 Inhibition of EGFR ligand induced HER3 phosphorylation CHOEGFR/HER3, TGFα construct IC50 (M) HER3MS00135 1.54E−10 (n = 3)HER3MS00137 5.078E−09 HER3MS00139 6.952E−09 HER3MS00141 5.636E−09HER3MS00147 no effect HER3MS00149 nd HER3MS00150 nd HER3MS00209 2.15E−09(n = 3) HER3MS00212 3.28E−09 (n = 3) HER3MS00213 1.37E−08 (n = 3)HER3MS00214 *4.02E−09 (n = 3)  HER3MS00215 2.95E−09 (n = 3) *partialinhibition

Example 23 Migration Blocking Capacity of the Multivalent SequenceOptimized Nanobodies with ALB11 HLE

The ability of formatted sequence optimized Nanobodies to inhibitHRG1-β1 dependent cell migration was assessed in the following assay.A431 cells (CRL 1555) were seeded in HTS Fluoroblok 96 well plateinserts (BD Falcon #351164) in the presence of Nanobodies (serialdilutions; starting at 666 nM). Media plus 500 nM HRG1-β1 extracellulardomain (R&D Systems, #377-HB) was added to the bottom wells. Migratedcells were stained with CalceinAM and fluorescence detected by platereader. Nanobodies HER3MS00135, HER3MS00212, and HER3MS00215 potentlyinhibited A431 cell migration (Table C-30). All tested formattedsequence optimized Nanbodies potently block ligand induced cellmigration.

TABLE C-30 Inhibition of HRG induced A431 cell migration construct IC50(M) HER3MS00135 2.09E−10 HER3MS00209 1.46E−08 HER3MS00212 4.46E−09HER3MS00213 7.09E−10 HER3MS00214 8.54E−11 HER3MS00215 9.75E−09

Example 24 HER3-Specificity of Multivalent Sequence OptimizedHER3-Specific Nanobodies with HLE

Off-target binding of multivalent HER3-specific Nanobodies with HLE wasassessed by measuring their binding capacity to Fc-HER1 (R&D Systems,344-ER), Fc-HER2 (R&D Systems, 1129-ER) and Fc-HER4 (R&D Systems,1131-ER) coated on ELISA plates. Serial dilutions of the Nanobodiesstarting at a concentration of 500 nM were tested and detection was doneusing the biotinylated ALB-specific Nanobody 02H05 and streptavidin-HRP(Dako, P0397). Binding to Fc-HER3 (R&D Systems, 348-RB) was used aspositive control for the HER3-specific Nanobodies. The polyclonalantibodies anti-HER1 (R&D Systems, AF231), anti-HER2 (R&D Systems,AF1129), anti-HER3 (R&D Systems, AF234) and anti-HER4 (R&D Systems,AF1131) were used as quality control of the HER coating on the plates.The results in FIGS. 3A to 3D show that multivalent sequence optimizedNanobodies bind to HER3 (FIG. 3B) but not to the other HER proteins(FIGS. 3A, 3C and 3D).

Example 25 Species Cross-Reactivity of Multivalent Sequence OptimizedHER3-Specific Nanobodies with HLE

The species cross-reactivity of multivalent sequence optimizedHER3-specific Nanobodies with HLE was analysed by measuring the bindingcapacity of these molecules to Chinese hamster ovary Flp-In cellstransfected with human HER3, mouse HER3 or cyno HER3 by FACS. Serialdilutions starting at a concentration of 1 μM were incubated with thecells and detection was done using the biotinylated ALB-specificNanobody 02H05 and streptavidin-PE (BD Pharmingen, 554061). The obtainedEC50 values were maximum 4.4-fold times different when comparing bindingto human HER3 with mouse HER3 or cyno HER3, suggesting cross-reactivitybetween human, mouse and cyno HER3 (Table C-31).

TABLE C-31 EC50-values of multivalent sequence optimized HER3-specificNanobodies with HLE in a FACS binding assay to human, mouse and cynoHER3 transfected CHO-FlpIn cells Human Mouse Cyno ID EC50 (M) CI95 (M)EC50 (M) CI95 (M) EC50 (M) CI95 (M) HER3MS00135 3.87E−09 3.228E−9 to1.09E−9 9.383E−10 to 1.37E−9 1.124E−9 to 4.640E−9 1.263E−9 1.674E−9HER3MS00212 8.29E−09 6.895E−9 to 4.14E−9 3.381E−9 to 4.36E−9 3.512E−9 to9.978E−9 5.081E−9 5.410E−9 HER3MS00215 8.85E−09 7.294E−9 to 6.30E−95.144E−9 to 6.42E−9 5.067E−9 to 1.074E−8 7.710E−9 8.128E−9 HER3MS002149.07E−09 7.451E−9 to 2.06E−9 1.704E−9 to 2.77E−9 2.213E−9 to 1.103E−82.497E−9 3.462E−9 HER3MS00213 9.21E−09 7.442E−9 to 1.18E−8 9.070E−9 to6.59E−9 5.358E−9 to 1.139E−8 1.527E−8 8.115E−9 HER3MS00209 6.43E−095.366E−9 to 3.92E−9 3.289E−9 to 4.10E−9 3.327E−9 to 7.709E−9 4.682E−95.040E−9

Example 26 Binding of Multivalent Sequence Optimized HER3-SpecificNanobodies with ALB11 HLE to Serum Albumin

Binding affinity of three multivalent sequence optimized HER3-specificNanobodies with ALB11 half life extension to human, mouse and cyno serumalbumin was determined by surface plasmon resonance using Biacore. Serumalbumin from human (Sigma #A3782), cyno (in house production) and mouse(Sigma, #A3559) were covalently bound to the sensor chip surface viaamine coupling using EDC/NHS for activation and ethanolamine HCl fordeactivation. Nanobodies were injected for 2 minutes at a flow rate of45 μl/min to allow binding to chip-bound antigen. Next, binding bufferwas sent over the chip at the same flow rate to allow spontaneousdissociation of bound Nanobody. The kinetic parameters kon-values (ka),koff-values (kd) and KD were calculated from the sensorgrams obtainedfor the different Nanobodies (Table C-32).

TABLE C-32 Kinetic parameters for multivalent sequence optimizedHER3-specific Nanobodies Human Cyno Mouse HER3MS00135 ka (1/Ms)1.68^(E)5  1.38^(E)5  1.31^(E)5  kd (1/Ms) 4.44^(E)−3 4.15^(E)−35.76^(E)−2 KD (nM) 26.5 30.1 439 HER3MS00212 ka (1/Ms) 1.64^(E)5 1.38^(E)5  1.19^(E)5  kd (1/Ms) 5.21^(E)−3 4.83^(E)−3 7.24^(E)−2 KD (nM)31.7 34.9 610 HER3MS00215 ka (1/Ms) 1.73^(E)5  1.47^(E)5  1.56^(E)5  kd(1/Ms) 4.57^(E)−3 4.29^(E)−3 5.93^(E)−2 KD (nM) 26.4 29.2 380

Example 27 Inhibition of pHER3 and Downstream Signaling in Human CancerCell Lines

Formatted sequence optimized Nanobodies potently inhibit pHER3 anddownstream signaling in human cancer cell lines as shown for MCF-7(Example 21 above) and others like BxPC3 a pancreatic cancer cell lineand BT-474 a breast cancer cell line.

17B05 and formatted Nanobodies including sequence optimized 17B05(HER3MS00119) as building block inhibit HER3 signaling in both EGFR/HER3driven (in BxPC3 cells) as well as HER2/HER3 (BT-474, Her2overexpressing cell line) cell lines.

BxPC3 (ATCC CRL-1687) and BT-47 (ATCC HTB-20) cells were serum starvedand pre-incubated with Nanobodies (serial dilutions, startingconcentration 167 nM) for 60 min at 37° C., 5% CO₂. Cells werestimulated with 100 ng/ml HRG1-β1 EGF domain (R&D Systems, #396-HB) for10 min, supernatants discarded and cells lysed in cold RIPA lysis buffer(1% NP-40 (Nonidet P40), 0.5% (V/V) Sodium-Deoxycholat, 0.1% (V/V) SDS,50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, protease inhibitorcocktail set III (Calbiochem), phosphatase inhibitor cocktail set II(Calbiochem)) for 45 min at 4° C. After centrifugation, 4× sample buffer(Biorad #161-0791) was added to the supernatants and samples analysed bySDS-PAGE (Criterion XT precast get, 4-12%, Bis Tris, Biorad #345-0125)followed by WB. Detection antibodies: pHER3Tyr1289 (CellSignaling#4791), pHER3 pTyr1197 (Cell Signaling#4561), pAkt (Ser473)(Cell Signaling#4060), pERK1/2 (Thr202/Thr204) (Cell Signaling#9101),HER3 (Millipore #05-390), Akt (Cell Signaling#9272), EKR1/2 (CellSignaling#4695).

The results are shown in FIGS. 5A and 5B, as well as FIGS. 6A to 6C.FIG. 5 shows the inhibition of pHER3 and downstream signalling in BT-474breast cancer cells by formatted sequence optimized Nanobodies: FIG.5A): lanes 1-6 Nanobody HER3MS00135 (0, 0.65, 2.7, 10, 42, 167 nM),lanes 7-12 Nanobody HER3MS00212 (0, 0.65, 2.7, 10, 42, 167 nM), lanes13-18 Nanobody HER3MS00215 (0, 0.65, 2.7, 10, 42, 167 nM), lane 19untreated, unstimulated cells. FIG. 5B): lanes 7-12 Nanobody HER3MS00119(0, 0.65, 2.7, 10, 42, 167 nM). FIG. 6 shows the inhibition of pHER3 anddownstream signalling in BxPC3 breast cancer cells by formatted sequenceoptimized Nanobodies: FIG. 6A): lanes 13-18 Nanobody HER3MS00 119 (0,0.65, 2.7, 10, 42, 167 nM), lanes 19-24 HER3MS00135 (0, 0.65, 2.7, 10,42, 167 nM). FIG. 6B) lanes 1-6 HER3MS00213 (0, 0.65, 2.7, 10, 42, 167nM), 7-12 HER3MS00214 (0, 0.65, 2.7, 10, 42, 167 nM). FIG. 6C): lanes1-6 HER3MS00209 (0, 0.65, 2.7, 10, 42, 167 nM), lanes 7-12 HER3MS00212(0, 0.65, 2.7, 10, 42, 167 nM), lanes 13-18 HER3MS00215 (0, 0.65, 2.7,10, 42, 167 nM), lane 19 untreated, unstimulated cells, lanes 20-24unrelated control Nanobody (0.65, 2.7, 10, 42, 167 nM).

Example 28 A549 In Vivo Model: Inhibition of Tumor Growth by FormattedSequence Optimized Nanobodies

To assess the efficacy of formatted sequence optimized Nanobodies, A549xenograft model of human lung cancer was established in nude mice andthe inhibition of tumor growth was assessed at multiple doses. T-Celldeficient nude/nude mice (6 weeks old female mice originated at NIH,outbread, albino background) were purchase from Charles RiverLaboratories (Wilmington, Mass.) for xenograft studies. A549 cells(ATCC, CCL-185) were grown in culture media (RPMI, 10% FBS; L-glutamine,37° C., 5% CO₂). Mice were injected with 5E+6 cells in 100 μl PBS on theright flank subcutaneously. Initial tumor growth and body weight wasmonitored. After 13 days tumors reached an average volume of 174 mm³ andmice were randomized into groups of 10. Mice were dosedintra-peritoneally every third day with 1 mg/kg, 3 mg/kg, or 10 mg/kg ofNanobodies HER3MS00135, HER3MS00212, or HER3MS00215, respectively. Alb11was used as control at 10 mg/kg. PBS was used as vehicle control.Treatment duration was 20 days. Tumor volume and body weight wererecorded twice per week. Percent body weight data were analyzed by OneWay ANOVA followed by Dunnett's post-hoc comparisons versus thecontrols. Tumor volume data were analyzed by Repeated Measures ANOVAfollowed by post-hoc Bonferrioni pair wise comparisons (α=0.05).

The median tumor volumes from each control and treatment group are shownin FIGS. 7A to 7C (for HER3MS00135, HER3MS00212 and HER3MS00215,respectively).

Administration of the Nanobodies resulted in significant dose dependenttumor growth inhibition of the human lung cancer xenograft model whencompared to tumors treated with PBS vehicle control or Alb11.

The invention claimed is:
 1. A protein or polypeptide comprising atleast two immunoglobulin single variable (ISV) domains that are eachdirected to and/or that can each specifically bind to human HER3 (SEQ IDNO: 1), in which each such ISV domain is independently: (a) capable ofinhibiting or blocking binding of HRG to HER-3; and/or (b) capable ofinhibiting or blocking heterodimerization of HER-3; and/or (c) capableof binding to domain II of HER-3, wherein said protein or polypeptidecomprises: (i) an ISV that is a 17B05-like sequence comprising: (A) aCDR1 comprising the amino acid sequence LNAMA (SEQ ID NO: 58); (B) aCDR2 comprising the amino acid sequence GIFGVGSTRYADSVKG (SEQ ID NO:88); and (C) a CDR3 comprising the amino acid sequence SSVTRGSSDY (SEQID NO: 118); and (ii) an ISV that is a 21F06-like sequence comprising:(A) a CDR1 comprising the amino acid sequence LNAMG (SEQ ID NO: 67); (B)a CDR2 comprising the amino acid sequence AIDWSDGNKDYADSVKG (SEQ ID NO:97) or AIDWSEGNKDYADSVKG (SEQ ID NO: 445); and (C) a CDR3 comprising theamino acid sequence DTPPWGPMIYIESYDS (SEQ ID NO: 127) orDTPPWGPLIYIESYDS (SEQ ID NO: 446).
 2. The protein or polypeptideaccording to claim 1, further functionalized to have an increasedhalf-life, for example by functionalisation and/or by including in theprotein or polypeptide a moiety or binding unit that increases thehalf-life of the construct.
 3. The protein or polypeptide according toclaim 2, wherein the functionalization comprises (i) including a moietyor binding unit; and/or (ii) including a peptide or binding unit thatcan bind to a serum protein.
 4. The protein or polypeptide according toclaim 1, comprising an ISV domain directed to human serum albumin. 5.The protein or polypeptide according to claim 1, selected from the groupconsisting of SEQ ID NO:282 and SEQ ID NO:199.
 6. A pharmaceuticalcomposition comprising a protein or polypeptide or ISV according toclaim
 1. 7. The protein or polypeptide according to claim 1, whereinsaid 17B05-like sequence is SEQ ID NO: 13, optionally, with one or moreof the amino acid residues at positions 11, 37, 44, 47, 83, 84, 103, 104and 108 according to the Kabat numbering chosen from the Hallmarkresidues mentioned in the following table: Position Human VH₃ HallmarkResidues 11 L, V L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I 37 V,I, F F, Y, V, L, A, H, S, IW, C, N, G, D, T, P 48 G E, Q, G, D, A, K, R,L, P, S, V, H, T, N, W, M, I 45 L L, R, P, H, F, G, Q, S, E, T, Y, C, I,D, V 47 W, Y F, L, W, G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D83 R, K R, K, T, E, Q, N, S, I, V, G, M, L, A, D, Y, H 84 A, T, D P, S,H, L, A, V, I, T F, D, R, Y, N, Q, G, E 103 W W, R, G, S, K, A, M, Y, L,F, T, N, V, Q, P, E, C 104 G G, A, S, T, D, P, N, E, C, L 108 L, M, T Q,L, R, P, E, K, S, T, M, A, H

wherein when positions 44, 45, 46, and 47 are G,L,E, and W,respectively, postion 108 is always Q in non-humanized V_(HH) sequencesthat also contain a W at position
 103. 8. The protein or polypeptideaccording to claim 2, wherein said 21F06-like sequence is SEQ ID NO: 22,optionally, with one or more of the amino acid residues at position 11,37, 44, 47, 83, 84, 103, 104 and 108 according to the Kabat numberingchosen from the Hallmark residues mentioned in the following table:Position Human VH₃ Hallmark Residues 11 L, V L, S, V, M, W, F, T, Q, E,A, R, G, K, Y, N, P, I 37 V, I, F F, Y, V, L, A, H, S, IW, C, N, G, D,T, P 48 G E, Q, G, D, A, K, R, L, P, S, V, H, T, N, W, M, I 45 L L, R,P, H, F, G, Q, S, E, T, Y, C, I, D, V 47 W, Y F, L, W, G, I, S, A, V, M,R, Y, E, P, T, C, H, K, Q, N, D 83 R, K R, K, T, E, Q, N, S, I, V, G, M,L, A, D, Y, H 84 A, T, D P, S, H, L, A, V, I, T F, D, R, Y, N, Q, G, E103 W W, R, G, S, K, A, M, Y, L, F, T, N, V, Q, P, E, C 104 G G, A, S,T, D, P, N, E, C, L 108 L, M, T Q, L, R, P, E, K, S, T, M, A, H

wherein when positions 44, 45, 46, and 47 are G,L,E, and W,respectively, position 108 is always Q in non-humanized V_(HH) sequencesthat also contain a W at position 103.